Recombinant polymerases for incorporation of protein shield nucleotide analogs

ABSTRACT

Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions, and/or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing or nucleic acid amplification. Such properties include enhanced performance with large nucleotide analogs, increased stability, increased readlength, and improved detection of modified bases, and can also include resistance to photodamage, enhanced metal ion coordination, reduced exonuclease activity, reduced reaction rates at one or more steps of the polymerase kinetic cycle, decreased branching fraction, altered cofactor selectivity, increased yield, increased accuracy, altered speed, increased cosolvent resistance, and the like. Also provided are nucleic acids which encode the polymerases with the aforementioned phenotypes, as well as methods of using such polymerases to make a DNA or to sequence a DNA template.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/842,467, filedDec. 14, 2017, which is a continuation of U.S. Ser. No. 15/188,672,filed Jun. 21, 2016, now U.S. Pat. No. 9,873,911, which is acontinuation of U.S. Ser. No. 14/042,318, filed Sep. 30, 2013, now U.S.Pat. No. 9,399,766, which claims priority to and benefit of thefollowing prior provisional patent applications: U.S. Ser. No.61/781,845, filed Mar. 14, 2013, entitled “RECOMBINANT POLYMERASES FORINCORPORATION OF PROTEIN SHIELD NUCLEOTIDE ANALOGS” by Satwik Kamtekarand Erik Miller, U.S. Ser. No. 61/764,971, filed Feb. 14, 2013, entitled“RECOMBINANT POLYMERASES FOR INCORPORATION OF PROTEIN SHIELD NUCLEOTIDEANALOGS” by Satwik Kamtekar and Erik Miller, and Ser. No. 61/708,469,filed Oct. 1, 2012, entitled “RECOMBINANT POLYMERASES WITH INCREASEDREADLENGTH AND STABILITY FOR SINGLE-MOLECULE SEQUENCING” by SatwikKamtekar and Erik Miller. Each of these applications is incorporatedherein by reference in its entirety for all purposes.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED BY U.S.P.T.O. eFS-WEB

The instant application contains a Sequence Listing which is beingsubmitted in computer readable form via the United States Patent andTrademark Office eFS-WEB system and which is hereby incorporated byreference in its entirety for all purposes. The txt file submittedherewith contains a 566 KB file(01016905_2020-05-22_SequenceListing.txt).

FIELD OF THE INVENTION

The invention relates to modified DNA polymerases for single moleculesequencing and nucleic acid amplification. The polymerases includerecombinant polymerases that exhibit enhanced utility with largenucleotide analogs. The invention also relates to methods for amplifyingnucleic acids and to methods for determining the sequence of nucleicacid molecules using such polymerases.

BACKGROUND OF THE INVENTION

DNA polymerases replicate the genomes of living organisms. In additionto this central role in biology, DNA polymerases are also ubiquitoustools of biotechnology. They are widely used, e.g., for reversetranscription, amplification, labeling, and sequencing, all centraltechnologies for a variety of applications such as nucleic acidsequencing, nucleic acid amplification, cloning, protein engineering,diagnostics, molecular medicine, and many other technologies.

Because of the importance of DNA polymerases, they have been extensivelystudied. This study has focused, e.g., on phylogenetic relationshipsamong polymerases, structure of polymerases, structure-function featuresof polymerases, and the role of polymerases in DNA replication and otherbasic biological processes, as well as ways of using DNA polymerases inbiotechnology. For a review of polymerases, see, e.g., Hübscher et al.(2002) “Eukaryotic DNA Polymerases” Annual Review of Biochemistry Vol.71: 133-163, Alba (2001) “Protein Family Review: Replicative DNAPolymerases” Genome Biology 2(1): reviews 3002.1-3002.4, Steitz (1999)“DNA polymerases: structural diversity and common mechanisms” J BiolChem 274:17395-17398, and Burgers et al. (2001) “Eukaryotic DNApolymerases: proposal for a revised nomenclature” J Biol Chem. 276(47):43487-90. Crystal structures have been solved for many polymerases,which often share a similar architecture. The basic mechanisms of actionfor many polymerases have been determined.

A fundamental application of DNA technology involves various labelingstrategies for labeling a DNA that is produced by a DNA polymerase. Thisis useful in DNA sequencing, microarray technology, SNP detection,cloning, PCR analysis, and many other applications. Labeling is oftenperformed in various post-synthesis hybridization or chemical labelingschemes, but DNA polymerases have also been used to directly incorporatevarious labeled nucleotides in a variety of applications, e.g., via nicktranslation, reverse transcription, random priming, amplification, thepolymerase chain reaction, etc. See, e.g., Giller et al. (2003)“Incorporation of reporter molecule-labeled nucleotides by DNApolymerases. I. Chemical synthesis of various reporter group-labeled2′-deoxyribonucleoside-5′-triphosphates” Nucleic Acids Res.31(10):2630-2635, Augustin et al. (2001) “Progress towardssingle-molecule sequencing: enzymatic synthesis ofnucleotide-specifically labeled DNA” J. Biotechnol. 86:289-301, Tonon etal. (2000) “Spectral karyotyping combined with locus-specific FISHsimultaneously defines genes and chromosomes involved in chromosomaltranslocations” Genes Chromosom. Cancer 27:418-423, Zhu and Waggoner(1997) “Molecular mechanism controlling the incorporation of fluorescentnucleotides into DNA by PCR” Cytometry, 28:206-211, Yu et al. (1994)“Cyanine dye dUTP analogs for enzymatic labeling of DNA probes” NucleicAcids Res. 22:3226-3232, Zhu et al. (1994) “Directly labeled DNA probesusing fluorescent nucleotides with different length linkers” NucleicAcids Res. 22:3418-3422, and Reid et al. (1992) “Simultaneousvisualization of seven different DNA probes by in situ hybridizationusing combinatorial fluorescence and digital imaging microscopy” Proc.Natl Acad. Sci. USA, 89:1388-1392.

DNA polymerase mutants have been identified that have a variety ofuseful properties, including altered nucleotide analog incorporationabilities relative to wild-type counterpart enzymes. For example,Vent^(A488L) DNA polymerase can incorporate certain non-standardnucleotides with a higher efficiency than native Vent DNA polymerase.See Gardner et al. (2004) “Comparative Kinetics of Nucleotide AnalogIncorporation by Vent DNA Polymerase” J. Biol. Chem. 279(12):11834-11842and Gardner and Jack “Determinants of nucleotide sugar recognition in anarchaeon DNA polymerase” Nucleic Acids Research 27(12):2545-2553. Thealtered residue in this mutant, A488, is predicted to be facing awayfrom the nucleotide binding site of the enzyme. The pattern of relaxedspecificity at this position roughly correlates with the size of thesubstituted amino acid side chain and affects incorporation by theenzyme of a variety of modified nucleotide sugars.

Additional modified polymerases, e.g., modified polymerases that displayimproved properties useful for single molecule sequencing (SMS) andother polymerase applications (e.g., DNA amplification, sequencing,labeling, detection, cloning, etc.), are desirable. The presentinvention provides new recombinant DNA polymerases with desirableproperties, including enhanced sequencing with large nucleotide analogs,increased readlength, and increased thermostability. Other exemplaryproperties include exonuclease deficiency, increased cosolventresistance, increased sensitivity to modified bases, altered cofactorselectivity, increased yield, increased resistance to photodamage,increased accuracy, increased speed, and the like. Also included aremethods of making and using such polymerases, as well as many otherfeatures that will become apparent upon a complete review of thefollowing.

SUMMARY OF THE INVENTION

Modified DNA polymerases can find use in such applications as, e.g.,single-molecule sequencing (SMS), genotyping analyses such as SNPgenotyping using single-base extension methods, sample preparation, andreal-time monitoring of amplification, e.g., RT-PCR. Among otheraspects, the invention provides compositions comprising recombinantpolymerases that comprise mutations which confer properties which can beparticularly desirable for these and other applications. Theseproperties can, e.g., improve performance with large nucleotide analogs,increase enzyme (and therefore assay) robustness, increase readlength,facilitate readout accuracy, or otherwise improve polymeraseperformance. Also provided by the invention are methods of generatingsuch modified polymerases and methods in which such polymerases can beused to, e.g., sequence a DNA template and/or make a DNA.

In one aspect, the invention provides compositions and sequencingsystems that include one or more large nucleotide analogs (for example,an analog that includes a protein or other moiety that shields thepolymerase from damage by one or more fluorescent dyes in the analog)and a polymerase modified to optimize usage of the large analog, e.g.,in single-molecule sequencing or another application. Accordingly, onegeneral class of embodiments provides a composition that comprises arecombinant DNA polymerase (e.g., a Φ29-type DNA polymerase) andoptionally a nucleotide analog. The recombinant polymerase comprises oneor more mutation that enhances performance with the analog, for example,one or more mutation that decreases interpulse distance, increases pulsewidth, reduces pausing, increases stability (e.g., free polymerase,binary complex, and/or or ternary complex stability), and/or increasesaccuracy, e.g., as compared to a corresponding wild-type or otherparental polymerase (e.g., a polymerase from which the modifiedrecombinant polymerase of the invention was derived, e.g., by mutation).

Accordingly, in one class of embodiments, the recombinant polymerasecomprises one or more mutation selected from the group consisting of anamino acid substitution at position Q99, an amino acid substitution atposition A134, an amino acid substitution at position K138, an aminoacid substitution at position V141, an amino acid substitution atposition L142, an amino acid substitution at position A256, an aminoacid substitution at position R306, an amino acid substitution atposition R308, an amino acid substitution at position K311, an aminoacid substitution at position T441, an amino acid substitution atposition E466, an amino acid substitution at position D476, an aminoacid substitution at position S487, an amino acid substitution atposition E508, an amino acid substitution at position L513, an aminoacid substitution at position D523, an amino acid substitution atposition I524, an amino acid substitution at position K536, an aminoacid substitution at position P558, an amino acid substitution atposition D570, and an amino acid substitution at position T571, whereinidentification of positions is relative to SEQ ID NO:1. For example, thepolymerase optionally comprises one or more mutation selected from thegroup consisting of an A256S substitution, an S487A substitution, aV141K substitution, an L142K substitution, a D476H substitution, anE508R substitution, a D523R substitution, a Q99P substitution, a Q99Ysubstitution, an R306Q substitution, an R308L substitution, a K311Esubstitution, a T441I substitution, a D570S substitution, a D570Tsubstitution, a D570E substitution, a D570M substitution, an A134Ssubstitution, an I524S substitution, an I524T substitution, an E466Ksubstitution, an L513K substitution, a T571V substitution, a T571Asubstitution, a P558A substitution, a P558F substitution, a K138Qsubstitution, a K138C substitution, a K138A substitution, a K536Qsubstitution, a K536T, and a K536E substitution, wherein identificationof positions is relative to wild-type Φ29 polymerase (SEQ ID NO:1).

The polymerase can also include mutations at additional positions. Forexample, the polymerase can include one or more mutation selected fromthe group consisting of an amino acid substitution at position 224, anamino acid substitution at position 239, an amino acid substitution atposition 253, an amino acid substitution at position 375, an amino acidsubstitution at position 437, an amino acid substitution at position484, an amino acid substitution at position 510, an amino acidsubstitution at position 512, and an amino acid substitution at position515, wherein identification of positions is relative to SEQ ID NO:1. Forexample, the polymerase optionally includes one or more mutationselected from the group consisting of a Y224K substitution, an E239Gsubstitution, an L253A substitution, an E375Y substitution, an A437Gsubstitution, an A437N substitution, an A484E substitution, a D510Ksubstitution, a K512Y substitution, and an E515Q substitution, whereinidentification of positions is relative to SEQ ID NO:1.

Optionally, the polymerase comprises mutations at two, three, four,five, six, seven, eight, nine, ten, or even eleven or more of thesepositions. For example, the polymerase can include a mutation atposition E375, a mutation at position K512, and a mutation at one ormore additional positions, e.g., as described herein. Similarly, forexample, the polymerase can comprise mutations at positions 375, 512,and 253, positions 375, 512, and 484 (e.g., E375Y, A484E, and K512Ysubstitutions), positions 253 and 484, positions 375, 512, 253, and 484,positions 375, 512, 253, 484, and 510, positions 253 and 437, positions239, 253, 375, 437, 484, 510, 512, and 515, or positions 224, 239, 253,375, 437, 484, 510, 512, and 515, and a mutation at one or moreadditional positions, e.g., as described herein, where identification ofpositions is relative to wild-type Φ29 polymerase (SEQ ID NO:1). Anumber of exemplary substitutions at these (and other) positions aredescribed herein.

As a few examples, the recombinant polymerase can comprise a combinationof mutations selected from the group consisting of aa) V141K, L142K,Y224K, E239G, V250I, L253A, A256S, R306Q, R308L, K311E, E375Y, A437G,T441I, A484E, S487A, E508R, D510K, K512Y, and E515Q; ab) Y224K, E239G,V250I, L253A, A256S, E375Y, A437G, A484E, S487A, D510K, K512Y, andE515Q; ac) V141K, L142K, Y224K, E239G, V250I, L253A, A256S, E375Y,A437G, T441I, A484E, S487A, E508R, D510K, K512Y, E515Q, and D570E; ad)A134S, Y224K, E239G, V250I, L253A, A256S, E375Y, A437G, A484E, S487A,D510K, K512Y, and E515Q; ae) Y224K, E239G, V250I, L253A, E375Y, A437G,A484E, E508R, D510K, K512Y, E515Q, and D570M; af) A134S, Y224K, E239G,V250I, L253A, E375Y, A437G, A484E, S487A, E508R, D510K, K512Y, E515Q,I524S, F526L, and D570E; ag) L142K, Y224K, E239G, V250I, L253A, E375Y,A437G, D476H, A484E, E508R, D510K, K512Y, L513K, E515Q, and D570E; ah)L142K, Y224K, E239G, V250I, L253A, R306Q, R308L, E375Y, A437G, D476H,A484E, E508R, D510K, K512Y, L513K, E515Q, and D570E; ai) L142K, Y224K,E239G, V250I, L253A, R306Q, R308L, E375Y, A437G, E466K, D476H, A484E,E508R, D510K, K512Y, L513K, E515Q, and D570E; aj) L142K, Y224K, E239G,V250I, L253A, R306Q, R308L, E375Y, A437G, E466K, D476H, A484E, E508R,D510K, K512Y, E515Q, and D570E; ak) L142K, Y224K, E239G, V250I, L253A,E375Y, A437G, E466K, D476H, A484E, E508R, D510K, K512Y, E515Q, andD570E; al) L142K, Y224K, E239G, V250I, L253A, E375Y, M429A, A437G,A484E, E508R, D510K, K512Y, E515Q, and D570E; am) A134S, L142K, Y224K,E239G, V250I, L253A, E375Y, A437G, A484E, S487A, E508R, D510K, K512Y,E515Q, I524S, F526L, and D570E; an) Y224K, E239G, V250I, L253A, E375Y,A437G, A484E, S487A, E508R, D510K, K512Y, E515Q, I524S, F526L, andD570M; ao) C106S, E239G, V250I, L253A, E375Y, A437G, A484E, E508R,D510K, K512Y, and E515Q; ap) L142K, Y224K, D235E, E239G, V250A, L253H,E375Y, A437G, A484E, E508R, D510K, K512Y, E515Q, and D570E; aq) Y224K,E239G, V250I, L253A, E375Y, A437G, A484E, E508R, D510K, K512Y, andE515Q; ar) V141K, L142K, Y224K, E239G, V250I, L253A, E375Y, A437G,A484E, E508R, D510K, K512Y, and E515Q; as) K135Q, L142K, Y224K, E239G,V250I, L253A, R306Q, R308L, E375Y, A437G, E466K, D476H, A484E, E508R,D510R, K512Y, E515Q, D570S, and T571V; at) K131E, K135Q, L142K, Y224K,E239G, V250I, L253A, E375Y, A437G, A484E, E508R, D510R, K512Y, E515Q,D523R, P558A, D570S, and T571V; au) A49E, C106S, K114R, K131E, K135Q,L142K, Y224K, E239G, V250I, L253A, Y369E, E375Y, A437G, D476H, A484E,E508R, D510K, K512Y, E515Q, D523R, D570S, and T571V; av) K135Q, K138Q,L142K, Y224K, E239G, V250I, L253A, R306Q, R308L, E375Y, A437G, E466K,D476H, A484E, E508R, D510K, K512Y, E515Q, I524T, P558A, D570S, andT571V; aw) K135Q, K138Q, L142K, Y224K, E239G, V250I, L253A, R306Q,R308L, E375Y, A437G, E466K, V475I, D476H, A484E, E508R, D510K, K512Y,E515Q, I524T, P558A, D570S, and T571V; ax) K135Q, K138Q, L142K, Y224K,E239G, V250I, L253A, R306Q, R308L, T368S, E375Y, A437G, E466K, D476H,A484E, E508R, D510K, K512Y, E515Q, I524T, P558A, D570S, and T571V; ay)K135Q, K138Q, L142K, Y224K, E239G, V250I, L253A, R306Q, R308L, T368S,E375Y, A437G, E466K, V475I, D476H, A484E, E508R, D510K, K512Y, E515Q,P558A, D570S, and T571V; az) L44A, K135Q, K138Q, L142K, Y224K, E239G,V250I, L253A, R306Q, R308L, E375Y, A437G, E466K, D476H, A484E, S487A,E508R, D510K, K512Y, E515Q, P558A, D570S, and T571V; ba) K135Q, L142K,Y224K, E239G, V250I, L253A, R306Q, R308L, T368S, E375Y, A437G, E466K,D476H, A484E, E508R, D510R, K512Y, E515Q, P558A, D570S, and T571V; bb)A49E, C106S, K114R, K135Q, L142K, Y224K, E239G, V250I, L253A, R306Q,R308L, E375Y, A437G, E466K, D476H, A484E, E508R, D510R, K512Y, E515Q,K536Q, K539Q, D570S, and T571V; bc) L44A, K135Q, K138Q, L142K, Y224K,E239G, V250I, L253A, R306Q, R308L, T368S, E375Y, A437G, E466K, D476H,A484E, E508R, D510K, K512Y, E515Q, P558A, D570S, and T571V; bd) L142K,Y224K, E239G, V250I, L253A, R306Q, R308L, E375Y, A437G, E466K, D476H,A484E, E508R, D510K, K512Y, E515Q, P558A, D570T, and T571A; be) K138C,L142K, Y224K, E239G, V250I, L253A, R306Q, R308L, E375Y, A437G, E466K,D476H, A484E, E508R, D510K, K512Y, E515Q, D570S, and T571V; bf) K138Q,L142K, Y224K, E239G, V250I, L253A, R306Q, R308L, E375Y, A437G, E466K,D476H, A484E, E508R, D510K, K512Y, E515Q, D570S, and T571V; bg) K135Q,L142K, Y224K, E239G, V250I, L253A, R306Q, R308L, E375Y, A437G, E466K,D476H, A484E, E508R, D510R, K512Y, E515Q, K539E, D570S, and T571V; bh)K131E, K135Q, L142K, Y224K, E239G, V250I, L253A, E375Y, A437G, D476H,A484E, E508R, D510K, K512Y, E515Q, D523R, D570S, and T571V; and bi)L142K, Y224K, E239G, V250I, L253A, R306Q, R308L, E375Y, A437G, E466K,D476H, A484E, E508R, D510K, K512Y, E515Q, and D570M; whereinidentification of positions is relative to SEQ ID NO:1. The recombinantpolymerase optionally comprises an amino acid sequence selected from thegroup consisting of: SEQ ID NOs:23-26, SEQ ID NOs:43-46, and SEQ IDNOs:67-128. Additional exemplary mutations and combinations of mutationsare described herein or can be formed from those disclosed herein, andpolymerases including such combinations are also features of theinvention.

The recombinant polymerase can be a modified recombinant Φ29 polymerase.Thus, in one class of embodiments, the recombinant polymerase is atleast 70% identical to wild-type Φ29 polymerase (SEQ ID NO:1), forexample, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or even at least 99% identical to wild-type Φ29 polymerase(SEQ ID NO:1). As another example, the recombinant polymerase can be amodified recombinant M2Y polymerase. Thus, in one class of embodiments,the recombinant polymerase is at least 70% identical to wild-type M2Ypolymerase (SEQ ID NO:2), for example, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or even at least 99% identical towild-type M2Y polymerase (SEQ ID NO:2). Optionally, the modifiedrecombinant M2Y polymerase comprises a glutamic acid residue at position175, an alanine residue at position 256, and/or a methionine residue atposition 506, wherein identification of positions is relative to SEQ IDNO:1. In other exemplary classes of embodiments, the recombinantpolymerase is a recombinant B103, GA-1, PZA, Φ15, BS32, Nf, G1, Cp-1,PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase.

The recombinant polymerase optionally comprises one or more exogenousfeatures, e.g., at the C-terminal and/or N-terminal region of thepolymerase, for example, a polyhistidine tag (e.g., a His10 tag) or abiotin ligase recognition sequence. As a few examples, the polymerasecan include a C-terminal polyhistidine tag, a C-terminal polyhistidinetag and biotin ligase recognition sequence, two tandem C-terminal biotinligase recognition sequences, or an N-terminal polyhistidine tag andbiotin ligase recognition sequence and a C-terminal polyhistidine tag.

The nucleotide analog included in the composition is generally largerthan the nucleotides typically used for nucleic acid synthesis andtypically includes a label, e.g., a fluorescent label. In one class ofembodiments, the analog has a molecular weight of at least 10,000, forexample, a molecular weight of at least 20,000, at least 30,000, atleast 40,000, at least 50,000, or even at least 60,000, at least100,000, at least 200,000, or at least 300,000 Da. In one class ofembodiments, the analog comprises a polypeptide moiety positionedbetween a dye moiety and a nucleotide component of the analog. Thepolypeptide moiety can be, e.g., a tetrameric biotin-binding protein,avidin, streptavidin, neutravidin, tamavidin, or another avidin protein,ubiquitin, or another polypeptide moiety (e.g., a polypeptide comprisingat least 60 amino acids, e.g., 60 to 1,000 amino acids, e.g., 80 to 600amino acids). In one class of embodiments, the analog comprises amultivalent central core element that comprises at least one fluorescentdye. The core is surrounded by multiple intermediate chemical groups, atleast one of which includes a shield element, and multiple terminalchemical groups, at least one of which includes a nucleotide. A numberof exemplary analogs are described hereinbelow.

In one class of embodiments, the composition includes a DNA template,and the polymerase incorporates the nucleotide analog into a copynucleic acid in response to the DNA template. The composition can bepresent in a DNA sequencing system, e.g., a zero-mode waveguide (ZMW).The recombinant polymerase can be immobilized on a surface, for example,on a surface of a zero-mode waveguide, preferably in an active form.

In one aspect, the invention provides methods of sequencing a DNAtemplate. In the methods, a reaction mixture that includes the DNAtemplate, a replication initiating moiety that complexes with or isintegral to the template, one or more nucleotides and/or nucleotideanalogs including at least one large analog (e.g., a protein shieldanalog or other shielded analog or an analog with a molecular weight ofat least 10,000), and a recombinant polymerase of the invention (e.g., arecombinant Φ29-type DNA polymerase) is provided. The polymerase iscapable of replicating at least a portion of the template using themoiety in a template-dependent polymerization reaction. The reactionmixture is subjected to a polymerization reaction in which therecombinant polymerase replicates at least a portion of the template ina template-dependent manner, whereby the one or more nucleotides and/ornucleotide analogs are incorporated into the resulting DNA. A timesequence of incorporation of the one or more nucleotides and/ornucleotide analogs into the resulting DNA is identified.

The nucleotide analogs used in the methods can comprise a first analogand a second analog (and optionally third, fourth, etc. analogs), eachof which comprise different fluorescent labels. The differentfluorescent labels can optionally be distinguished from one anotherduring the step in which a time sequence of incorporation is identified.Optionally, subjecting the reaction mixture to a polymerization reactionand identifying a time sequence of incorporation are performed in a zeromode waveguide. Essentially all of the features noted for thecompositions herein apply to these methods as well, as relevant.

In a related aspect, the invention provides methods of making a DNA. Inthe methods, a reaction mixture is provided that includes a template, areplication initiating moiety that complexes with or is integral to thetemplate, one or more nucleotides and/or nucleotide analogs including atleast one large analog (e.g., a protein shield analog or other shieldedanalog or an analog with a molecular weight of at least 10,000), and arecombinant polymerase of the invention (e.g., a recombinant Φ29-typeDNA polymerase). The polymerase is capable of replicating at least aportion of the template using the moiety in a template-dependentpolymerase reaction. The mixture is reacted such that the polymerasereplicates at least a portion of the template in a template-dependentmanner, whereby the one or more nucleotides and/or nucleotide analogsare incorporated into the resulting DNA. The reaction mixture isoptionally reacted in a zero mode waveguide. The methods optionallyinclude detecting incorporation of at least one of the nucleotidesand/or nucleotide analogs. Essentially all of the features noted for thecompositions herein apply to these methods as well, as relevant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents an alignment between the amino acid sequences ofwild-type M2Y polymerase (SEQ ID NO:2) and wild-type Φ29 polymerase (SEQID NO:1).

FIG. 2 depicts the structure of A488dA4P.

FIGS. 3A-3B schematically illustrate an exemplary single moleculesequencing by incorporation process in which the compositions of theinvention provide particular advantages.

FIG. 4 presents a fluorescence time trace for a ZMW, showing pulsesrepresenting incorporation of different nucleotide analogs. The insetschematically illustrates the catalytic cycle for polymerase-mediatedextension; the box indicates the portion of the catalytic cycle thatcorresponds to the pulse when sequencing is performed withphosphate-labeled nucleotide analogs.

FIG. 5A schematically illustrates a thermal inactivation assay. Thegapped substrate is listed as SEQ ID NOs: 64 and 65 and the extendedstrand as SEQ ID NO:66. FIG. 5B presents thermal inactivation profilesfor six recombinant Φ29 polymerases. FIG. 5C presents a bar graphillustrating that addition of a D570E substitution to a variety ofrecombinant Φ29 polymerases increases their readlength. Binary IT50values for the polymerases are also shown, over the graph.

FIG. 6 shows a view in the vicinity of residue M429 of Φ29 polymerase.Residues from the fingers domain are shown as sticks, and the locationof the incoming nucleotide is indicated by an arrow.

FIG. 7 provides exemplary polymerase mutations and combinations thereofin accordance with the invention. Positions of the mutations areidentified relative to a wild-type Φ29 DNA polymerase (SEQ ID NO:1)where the name of the polymerase includes “Phi29” or relative to awild-type M2Y polymerase (SEQ ID NO:2) where the name of the polymeraseincludes “M2.”

FIG. 8 provides exemplary polymerase mutations and combinations thereofin accordance with the invention. Positions of the mutations areidentified relative to a wild-type Φ29 DNA polymerase (SEQ ID NO:1)where the name of the polymerase includes “Phi29” or relative to awild-type M2Y polymerase (SEQ ID NO:2) where the name of the polymeraseincludes “M2.”

FIG. 9 provides exemplary polymerase mutations and combinations thereofin accordance with the invention. Positions of the mutations areidentified relative to a wild-type Φ29 DNA polymerase (SEQ ID NO:1)where the name of the polymerase includes “Phi29” or relative to awild-type M2Y polymerase (SEQ ID NO:2) where the name of the polymeraseincludes “M2.”

Schematic figures are not necessarily to scale.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. The following definitionssupplement those in the art and are directed to the current applicationand are not to be imputed to any related or unrelated case, e.g., to anycommonly owned patent or application. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice for testing of the present invention, the preferred materialsand methods are described herein. Accordingly, the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a protein”includes a plurality of proteins; reference to “a cell” includesmixtures of cells, and the like.

The term “about” as used herein indicates the value of a given quantityvaries by +/−10% of the value, or optionally +/−5% of the value, or insome embodiments, by +/−1% of the value so described.

The term “nucleic acid” or “polynucleotide” encompasses any physicalstring of monomer units that can be corresponded to a string ofnucleotides, including a polymer of nucleotides (e.g., a typical DNA orRNA polymer), PNAs, modified oligonucleotides (e.g., oligonucleotidescomprising nucleotides that are not typical to biological RNA or DNA,such as 2′-O-methylated oligonucleotides), and the like. A nucleic acidcan be e.g., single-stranded or double-stranded. Unless otherwiseindicated, a particular nucleic acid sequence of this inventionencompasses complementary sequences, in addition to the sequenceexplicitly indicated.

A “polypeptide” is a polymer comprising two or more amino acid residues(e.g., a peptide or a protein). The polymer can additionally comprisenon-amino acid elements such as labels, quenchers, blocking groups, orthe like and can optionally comprise modifications such asglycosylation, biotinylation, or the like. The amino acid residues ofthe polypeptide can be natural or non-natural and can be unsubstituted,unmodified, substituted or modified.

An “amino acid sequence” is a polymer of amino acid residues (a protein,polypeptide, etc.) or a character string representing an amino acidpolymer, depending on context.

A “polynucleotide sequence” or “nucleotide sequence” is a polymer ofnucleotides (an oligonucleotide, a DNA, a nucleic acid, etc.) or acharacter string representing a nucleotide polymer, depending oncontext. From any specified polynucleotide sequence, either the givennucleic acid or the complementary polynucleotide sequence (e.g., thecomplementary nucleic acid) can be determined.

Numbering of a given amino acid or nucleotide polymer “corresponds tonumbering of” or is “relative to” a selected amino acid polymer ornucleic acid when the position of any given polymer component (aminoacid residue, incorporated nucleotide, etc.) is designated by referenceto the same residue position in the selected amino acid or nucleotidepolymer, rather than by the actual position of the component in thegiven polymer. Similarly, identification of a given position within agiven amino acid or nucleotide polymer is “relative to” a selected aminoacid or nucleotide polymer when the position of any given polymercomponent (amino acid residue, incorporated nucleotide, etc.) isdesignated by reference to the residue name and position in the selectedamino acid or nucleotide polymer, rather than by the actual name andposition of the component in the given polymer. Correspondence ofpositions is typically determined by aligning the relevant amino acid orpolynucleotide sequences. For example, residue K221 of wild-type M2Ypolymerase (SEQ ID NO:2) is identified as position Y224 relative towild-type Φ29 polymerase (SEQ ID NO:1); see, e.g., the alignment shownin FIG. 1. Similarly, residue L138 of wild-type M2Y polymerase (SEQ IDNO:2) is identified as position V141 relative to wild-type Φ29polymerase (SEQ ID NO:1), and an L138K substitution in the M2Ypolymerase is thus identified as a V141K substitution relative to SEQ IDNO:1. Amino acid positions herein are generally identified relative toSEQ ID NO:1 unless explicitly indicated otherwise.

The term “recombinant” indicates that the material (e.g., a nucleic acidor a protein) has been artificially or synthetically (non-naturally)altered by human intervention. The alteration can be performed on thematerial within, or removed from, its natural environment or state. Forexample, a “recombinant nucleic acid” is one that is made by recombiningnucleic acids, e.g., during cloning, DNA shuffling or other procedures,or by chemical or other mutagenesis; a “recombinant polypeptide” or“recombinant protein” is, e.g., a polypeptide or protein which isproduced by expression of a recombinant nucleic acid.

A “Φ29-type DNA polymerase” (or “phi29-type DNA polymerase”) is a DNApolymerase from the Φ29 phage or from one of the related phages that,like Φ29, contain a terminal protein used in the initiation of DNAreplication. Φ29-type DNA polymerases are homologous to the Φ29 DNApolymerase (e.g., as listed in SEQ ID NO:1); examples include the B103,GA-1, PZA, Φ15, BS32, M2Y (also known as M2), Nf, G1, Cp-1, PRD1, PZE,SF5, Cp-5, Cp-7, PR4, PR5, PR722, L17, and AV-1 DNA polymerases, as wellas chimeras thereof. A modified recombinant Φ29-type DNA polymeraseincludes one or more mutations relative to naturally-occurring wild-typeΦ29-type DNA polymerases, for example, one or more mutations thatincrease stability, increase readlength, alter interaction with and/orincorporation of nucleotide analogs, enhance accuracy, increasephototolerance, and/or alter another polymerase property, and mayinclude additional alterations or modifications over the wild-typeΦ29-type DNA polymerase, such as one or more deletions, insertions,and/or fusions of additional peptide or protein sequences (e.g., forimmobilizing the polymerase on a surface or otherwise tagging thepolymerase enzyme).

A variety of additional terms are defined or otherwise characterizedherein.

DETAILED DESCRIPTION

Among other aspects, the present invention provides new recombinantpolymerases. A recombinant polymerase of the invention, e.g., arecombinant Φ29-type DNA polymerase, typically includes one or moremutations (e.g., amino acid substitutions, deletions, or insertions) ascompared to a reference polymerase, e.g., a wild-type Φ29-typepolymerase. Depending on the particular mutation or combination ofmutations, the polymerase exhibits one or more properties that find usein, e.g., single molecule sequencing applications or nucleic acidamplification. Such polymerases incorporate nucleotides and/ornucleotide analogs, for example, dye labeled phosphate labeled analogs,into a growing template copy during DNA amplification. These polymerasesare modified such that they have one or more desirable properties, forexample, improved sequencing performance with large nucleotide analogs,increased readlength, increased thermostability, increased resistance tophotodamage, decreased branching fraction formation when incorporatingthe relevant analogs, improved DNA-polymerase stability or processivity,increased cosolvent resistance, reduced exonuclease activity, increasedyield, altered cofactor selectivity, improved accuracy, increased ordecreased speed, and/or altered kinetic properties (e.g., a reduction inthe rate of one or more steps of the polymerase kinetic cycle, resultingfrom, e.g., enhanced interaction of the polymerase with nucleotideanalog, enhanced metal coordination, etc.) as compared to acorresponding wild-type or other parental polymerase (e.g., a polymerasefrom which the modified recombinant polymerase of the invention wasderived, e.g., by mutation), as well as other features that will becomeapparent upon a complete review of the present disclosure. Thepolymerases of the invention can also include any of the additionalfeatures for improved specificity, processivity, retention time, surfacestability, analog incorporation, and/or the like noted herein. Thepolymerases can include one or more exogenous or heterologous features,e.g., at the N- and/or C-terminal regions of the polymerase. Suchfeatures find use not only for purification of the recombinantpolymerase and/or immobilization of the polymerase to a substrate, butcan also alter one or more properties of the polymerase.

These new polymerases are particularly well suited to DNA amplificationand/or sequencing applications, particularly sequencing protocols thatinclude detection in real time of the incorporation of labeled analogsinto DNA amplicons. As a few examples, increased readlength can producelonger sequence reads that facilitate scaffolding of sequences obtainedfrom shorter reads during genome assembly, increased phototolerance canprolong useful life of the polymerase under assay conditions, andaltered rates, reduced or eliminated exonuclease activity, decreasedbranching fraction, improved complex stability, altered metal cofactorselectivity, or the like can facilitate discrimination of nucleotideincorporation events from non-incorporation events such as transientbinding of a mismatched nucleotide in the active site of the complex,improve processivity, and/or facilitate detection of incorporationevents.

Exemplary polymerases include a recombinant Φ29-type DNA polymerase thatcomprises a mutation at one or more positions selected from the groupconsisting of C11, L44, A49, Q99, C106, K114, L123, K131, A134, K135,K138, V141, L142, Y148, E175, Y224, D235, E239, V250, L253, A256, C290,R306, R308, K311, K337, Y369, E375, M429, A437, T441, C448, C455, E466,V475, D476, A484, H485, 5487, E508, D510, K512, L513, E515, D523, I524,F526, K536, K539, P558, D570, and T571, where identification ofpositions is relative to wild-type Φ29 polymerase (SEQ ID NO:1).Optionally, the polymerase comprises mutations at two, three, four,five, six, seven, eight, nine, ten, or even eleven or more of thesepositions. For example, the polymerase can include a mutation atposition E375, a mutation at position K512, and a mutation at one ormore additional positions, e.g., as described herein. Similarly, forexample, the polymerase can comprise mutations at positions 375, 512,and 253, positions 375, 512, and 484, positions 253 and 484, positions375, 512, 253, and 484, positions 375, 512, 253, 484, and 510, positions253 and 437, positions 253 and 250, positions 253, 437, and 250,positions 148 and 570, positions 239, 253, 375, 437, 484, 510, 512, and515, positions 224, 239, 253, 375, 437, 484, 510, 512, and 515,positions 148, 224, 239, 253, 375, 437, 484, 510, 512, and 515, orpositions 131, 224, 239, 253, 375, 437, 484, 510, 512, and 515, and amutation at one or more additional positions, e.g., as described herein,where identification of positions is relative to wild-type Φ29polymerase (SEQ ID NO:1). A number of exemplary substitutions at these(and other) positions are described herein.

As a few examples, a mutation at E375 can comprise an amino acidsubstitution selected from the group consisting of E375Y (i.e., atyrosine residue is present at position E375 where identification ofpositions is relative to SEQ ID NO:1), E375F, E375R, E375Q, E375H,E375L, E375A, E375K, E375S, E375T, E375C, E375G, and E375N; a mutationat position K512 can comprise an amino acid substitution selected fromthe group consisting of K512Y, K512F, K512I, K512M, K512C, K512E, K512G,K512H, K512N, K512Q, K512R, K512V, and K512H; a mutation at positionL253 can comprise an amino acid substitution selected from the groupconsisting of L253A, L253H, L253S, and L253C; a mutation at positionA484 can comprise an A484E substitution; and/or a mutation at positionD510 can comprise a D510K or D510S substitution. Other exemplarysubstitutions include, e.g., C11A, L44A, A49E, Q99P, Q99I, Q99W, Q99Y,C106S, K114R, L123K, L123Y, L123R, L123Q, L123A, K131E, A134S, K135Q,K135S, K138Q, K138C, K138A, V141K, L142K, Y148I, Y224K, D235E, E239G,V250A, V250I, A256S, C290F, R306Q, R308L, K311E, K337C, K337Q, Y369E,M429A, A437G, A437N, T441I, C448V, C455A, E466K, V475I, D476H, H485Q,S487A, E508K, E508R, L513K, E515Q, D523R, I524S, I524T, F526L, K536Q,K536T, K536E, K539Q, K539E, P558A, P558F, D570E, D570N, D570S, D570T,D570H, D570L, D570M, D570V, D570I, D570W, D570Y, D570F, D570G, D570Q,D570K, D570R, D570P, D570A, D570C, T571V, and T571A; additionalsubstitutions are described herein.

The polymerase mutations and mutational strategies noted herein can becombined with each other and with essentially any other availablemutations and mutational strategies to confer additional improvementsin, e.g., nucleotide analog specificity, enzyme processivity, improvedretention time of labeled nucleotides in polymerase-DNA-nucleotidecomplexes, phototolerance, and the like. For example, the mutations andmutational strategies herein can be combined with those taught in, e.g.,WO 2007/076057 “Polymerases for Nucleotide Analogue Incorporation” byHanzel et al., WO 2008/051530 “Polymerase Enzymes and Reagents forEnhanced Nucleic Acid Sequencing” by Rank et al., US patent applicationpublication 2010-0075332 “Engineering Polymerases and ReactionConditions for Modified Incorporation Properties” by Pranav Patel etal., US patent application publication 2010-0093555 “Enzymes Resistantto Photodamage” by Keith Bjornson et al., US patent applicationpublication 2010-0112645 “Generation of Modified Polymerases forImproved Accuracy in Single Molecule Sequencing” by Sonya Clark et al.,US patent application publication 2011-0189659 “Generation of ModifiedPolymerases for Improved Accuracy in Single Molecule Sequencing” bySonya Clark et al., US patent application publication 2012-0034602“Recombinant Polymerases For Improved Single Molecule Sequencing” byRobin Emig et al., and U.S. patent application Ser. No. 13/756,113 filedJan. 31, 2013 by Satwik Kamtekar et al. and entitled “RecombinantPolymerases with Increased Phototolerance.” This combination ofmutations/mutational strategies can be used to impart severalsimultaneous improvements to a polymerase (e.g., enhanced utility withlarge analogs, increased readlength, increased phototolerance, decreasedbranching fraction formation, improved specificity, improvedprocessivity, altered rates, improved retention time, improved stabilityof the closed complex, tolerance for a particular metal cofactor, etc.).In addition, polymerases can be further modified forapplication-specific reasons, such as to improve activity of the enzymewhen bound to a surface, as taught, e.g., in WO 2007/075987 “ActiveSurface Coupled Polymerases” by Hanzel et al. and WO 2007/075873“Protein Engineering Strategies to Optimize Activity of Surface AttachedProteins” by Hanzel et al., or to include purification or handling tagsas is taught in the cited references and as is common in the art.Similarly, the modified polymerases described herein can be employed incombination with other strategies to improve polymerase performance, forexample, reaction conditions for controlling polymerase rate constantssuch as taught in US patent application publication US 2009-0286245entitled “Two slow-step polymerase enzyme systems and methods.”

Also taught are approaches for modifying polymerases to enhance one ormore properties exhibited by the polymerases or to confer an additionalproperty not provided by a starting combination of mutations. Forexample, provided below are approaches for obtaining polymerases withenhanced utility with large nucleotide analogs, increased readlength,increased thermostability, and improved ability to detect basemodifications.

DNA Polymerases

DNA polymerases that can be modified to have increased readlength,greater thermostability, and/or other desirable properties as describedherein are generally available. DNA polymerases are sometimes classifiedinto six main groups based upon various phylogenetic relationships,e.g., with E. coli Pol I (class A), E. coli Pol II (class B), E. coliPol III (class C), Euryarchaeotic Pol II (class D), human Pol beta(class X), and E. coli UmuC/DinB and eukaryotic RAD30/xerodermapigmentosum variant (class Y). For a review of recent nomenclature, see,e.g., Burgers et al. (2001) “Eukaryotic DNA polymerases: proposal for arevised nomenclature” J Biol Chem. 276(47):43487-90. For a review ofpolymerases, see, e.g., Hübscher et al. (2002) “Eukaryotic DNAPolymerases” Annual Review of Biochemistry Vol. 71: 133-163; Alba (2001)“Protein Family Review: Replicative DNA Polymerases” Genome Biology2(1):reviews 3002.1-3002.4; and Steitz (1999) “DNA polymerases:structural diversity and common mechanisms” J Biol Chem 274:17395-17398.The basic mechanisms of action for many polymerases have beendetermined. The sequences of literally hundreds of polymerases arepublicly available, and the crystal structures for many of these havebeen determined or can be inferred based upon similarity to solvedcrystal structures for homologous polymerases. For example, the crystalstructure of Φ29, a preferred type of parental enzyme to be modifiedaccording to the invention, is available.

Many such polymerases that are suitable for modification are available,e.g., for use in sequencing, labeling, and amplification technologies.For example, human DNA Polymerase Beta is available from R&D systems.DNA polymerase I is available from Epicenter, GE Health Care,Invitrogen, New England Biolabs, Promega, Roche Applied Science, SigmaAldrich, and many others. The Klenow fragment of DNA Polymerase I isavailable in both recombinant and protease digested versions, from,e.g., Ambion, Chimerx, eEnzyme LLC, GE Health Care, Invitrogen, NewEngland Biolabs, Promega, Roche Applied Science, Sigma Aldrich and manyothers. Φ29 DNA polymerase is available from e.g., Epicentre. Poly Apolymerase, reverse transcriptase, Sequenase, SP6 DNA polymerase, T4 DNApolymerase, T7 DNA polymerase, and a variety of thermostable DNApolymerases (Taq, hot start, titanium Taq, etc.) are available from avariety of these and other sources. Recent commercial DNA polymerasesinclude Phusion™ High-Fidelity DNA Polymerase, available from NewEngland Biolabs; GoTaq® Flexi DNA Polymerase, available from Promega;RepliPHI™ Φ29 DNA Polymerase, available from Epicentre Biotechnologies;PfuUltra™ Hotstart DNA Polymerase, available from Stratagene; KOD HiFiDNA Polymerase, available from Novagen; and many others.Biocompare(dot)com provides comparisons of many different commerciallyavailable polymerases.

DNA polymerases that are preferred substrates for mutation to enhanceperformance with large nucleotide analogs, increase readlength, improvethermostability, improve detection of base modifications, increasephototolerance, reduce reaction rates, reduce or eliminate exonucleaseactivity, alter metal cofactor selectivity, and/or alter one or moreother property described herein include Taq polymerases, exonucleasedeficient Taq polymerases, E. coli DNA Polymerase 1, Klenow fragment,reverse transcriptases, Φ29 related polymerases including wild type Φ29polymerase and derivatives of such polymerases such as exonucleasedeficient forms, T7 DNA polymerase, T5 DNA polymerase, RB69 polymerase,etc.

In one aspect, the polymerase that is modified is a Φ29-type DNApolymerase. For example, the modified recombinant DNA polymerase can behomologous to a wild-type or exonuclease deficient Φ29 DNA polymerase,e.g., as described in U.S. Pat. No. 5,001,050, 5,198,543, or 5,576,204.Similarly, the modified recombinant DNA polymerase can be homologous toanother Φ29-type DNA polymerase, such as B103, GA-1, PZA, Φ15, BS32, M2Y(also known as M2), Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5,PR722, L17, AV-1, D21, or the like. For nomenclature, see also, Meijeret al. (2001) “Φ29 Family of Phages” Microbiology and Molecular BiologyReviews, 65(2):261-287. See, e.g., SEQ ID NO:1 for the amino acidsequence of wild-type Φ29 polymerase, SEQ ID NO:2 for the amino acidsequence of wild-type M2Y polymerase, SEQ ID NO:3 for the amino acidsequence of wild-type B103 polymerase, SEQ ID NO:4 for the amino acidsequence of wild-type GA-1 polymerase, SEQ ID NO:5 for the amino acidsequence of wild-type AV-1 polymerase, and SEQ ID NO:6 for the aminoacid sequence of wild-type CP-1 polymerase.

In addition to wild-type polymerases, chimeric polymerases made from amosaic of different sources can be used. For example, Φ29-typepolymerases made by taking sequences from more than one parentalpolymerase into account can be used as a starting point for mutation toproduce the polymerases of the invention. Chimeras can be produced,e.g., using consideration of similarity regions between the polymerasesto define consensus sequences that are used in the chimera, or usinggene shuffling technologies in which multiple Φ29-related polymerasesare randomly or semi-randomly shuffled via available gene shufflingtechniques (e.g., via “family gene shuffling”; see Crameri et al. (1998)“DNA shuffling of a family of genes from diverse species acceleratesdirected evolution” Nature 391:288-291; Clackson et al. (1991) “Makingantibody fragments using phage display libraries” Nature 352:624-628;Gibbs et al. (2001) “Degenerate oligonucleotide gene shuffling (DOGS): amethod for enhancing the frequency of recombination with familyshuffling” Gene 271:13-20; and Hiraga and Arnold (2003) “General methodfor sequence-independent site-directed chimeragenesis: J. Mol. Biol.330:287-296). In these methods, the recombination points can bepredetermined such that the gene fragments assemble in the correctorder. However, the combinations, e.g., chimeras, can be formed atrandom. For example, using methods described in Clarkson et al., fivegene chimeras, e.g., comprising segments of a Phi29 polymerase, a PZApolymerase, a M2 polymerase, a B103 polymerase, and a GA-1 polymerase,can be generated. Appropriate mutations to enhance performance withlarge analogs, increase readlength, improve thermostability, and/oralter another desirable property as described herein can be introducedinto the chimeras.

Available DNA polymerase enzymes have also been modified in any of avariety of ways, e.g., to reduce or eliminate exonuclease activities(many native DNA polymerases have a proof-reading exonuclease functionthat interferes with, e.g., sequencing applications), to simplifyproduction by making protease digested enzyme fragments such as theKlenow fragment recombinant, etc. As noted, polymerases have also beenmodified to confer improvements in specificity, processivity, andretention time of labeled nucleotides in polymerase-DNA-nucleotidecomplexes (e.g., WO 2007/076057 “Polymerases for Nucleotide AnalogueIncorporation” by Hanzel et al. and WO 2008/051530 “Polymerase Enzymesand Reagents for Enhanced Nucleic Acid Sequencing” by Rank et al.), toalter branching fraction and translocation (e.g., US patent applicationpublication 2010-0075332 by Pranav Patel et al. entitled “EngineeringPolymerases and Reaction Conditions for Modified IncorporationProperties”), to increase photostability (e.g., US patent applicationpublication 2010-0093555 “Enzymes Resistant to Photodamage” by KeithBjornson et al. and U.S. patent application Ser. No. 13/756,113 filedJan. 31, 2013 by Satwik Kamtekar et al. and entitled “RecombinantPolymerases with Increased Phototolerance”), to slow one or morecatalytic steps during the polymerase kinetic cycle, increase closedcomplex stability, decrease branching fraction, alter cofactorselectivity, and increase yield, thermostability, accuracy, speed, andreadlength (e.g., US patent application publication 2010-0112645“Generation of Modified Polymerases for Improved Accuracy in SingleMolecule Sequencing” by Sonya Clark et al., US patent applicationpublication 2011-0189659 “Generation of Modified Polymerases forImproved Accuracy in Single Molecule Sequencing” by Sonya Clark et al.,and US patent application publication 2012-0034602 “RecombinantPolymerases For Improved Single Molecule Sequencing” by Robin Emig etal.), and to improve surface-immobilized enzyme activities (e.g., WO2007/075987 Active Surface Coupled Polymerases by Hanzel et al. and WO2007/075873 Protein Engineering Strategies to Optimize Activity ofSurface Attached Proteins by Hanzel et al.). Any of these availablepolymerases can be modified in accordance with the invention.

Nucleotide Analogs

As discussed, various polymerases of the invention can incorporate oneor more nucleotide analogs into a growing oligonucleotide chain. Uponincorporation, the analog can leave a residue that is the same as ordifferent than a natural nucleotide in the growing oligonucleotide (thepolymerase can incorporate any non-standard moiety of the analog, or cancleave it off during incorporation into the oligonucleotide). A“nucleotide analog” herein is a compound, that, in a particularapplication, functions in a manner similar or analogous to a naturallyoccurring nucleoside triphosphate (a “nucleotide”), and does nototherwise denote any particular structure. A nucleotide analog is ananalog other than a standard naturally occurring nucleotide, i.e., otherthan A, G, C, T, or U, though upon incorporation into theoligonucleotide, the resulting residue in the oligonucleotide can be thesame as (or different from) an A, G, C, T, or U residue.

In one useful aspect of the invention, nucleotide analogs can also bemodified to achieve any of the improved properties desired. For example,various linkers or other substituents can be incorporated into analogsthat have the effect of reducing branching fraction, improvingprocessivity, or altering rates. Modifications to the analogs caninclude extending the phosphate chains, e.g., to include a tetra-,penta-, hexa- or heptaphosphate group, and/or adding chemical linkers toextend the distance between the nucleotide base and the dye molecule,e.g., a fluorescent dye molecule. Substitution of one or morenon-bridging oxygen in the polyphosphate, for example with S or BH₃, canchange the polymerase reaction kinetics, e.g., to achieve a systemhaving two slow steps as described hereinbelow. Optionally, one or more,two or more, three or more, or four or more non-bridging oxygen atoms inthe polyphosphate group of the analog has an S substituted for an O.While not being bound by theory, it is believed that the properties ofthe nucleotide, such as the metal chelation properties,electronegativity, or steric properties, can be altered by substitutionof the non-bridging oxygen(s).

Many nucleotide analogs are available and can be incorporated by thepolymerases of the invention. These include analog structures with coresimilarity to naturally occurring nucleotides, such as those thatcomprise one or more substituent on a phosphate, sugar, or base moietyof the nucleoside or nucleotide relative to a naturally occurringnucleoside or nucleotide. In one embodiment, the nucleotide analogincludes three phosphate containing groups; for example, the analog canbe a labeled nucleoside triphosphate analog and/or an α-thiophosphatenucleotide analog having three phosphate groups. In one embodiment, anucleotide analog can include one or more extra phosphate containinggroups, relative to a nucleoside triphosphate. For example, a variety ofnucleotide analogs that comprise, e.g., from 4-6 or more phosphates aredescribed in detail in US patent application publication 2007-0072196,incorporated herein by reference in its entirety for all purposes. Otherexemplary useful analogs, including tetraphosphate and pentaphosphateanalogs, are described in U.S. Pat. No. 7,041,812, incorporated hereinby reference in its entirety for all purposes.

For example, the analog can include a labeled compound of the formula:

wherein B is a nucleobase (and optionally includes a label); S isselected from a sugar moiety, an acyclic moiety or a carbocyclic moiety(and optionally includes a label); L is an optional detectable label; R₁is selected from O and S; R₂, R₃ and R₄ are independently selected fromO, NH, S, methylene, substituted methylene, C(O), C(CH₂), CNH₂, CH₂CH₂,and C(OH)CH₂R where R is 4-pyridine or 1-imidazole, provided that R₄ mayadditionally be selected from

R₅, R₆, R₇, R₈, R₁₁ and R₁₃ are, when present, each independentlyselected from O, BH₃, and S; and R₉, R₁₀ and R₁₂ are independentlyselected from O, NH, S, methylene, substituted methylene, CNH₂, CH₂CH₂,and C(OH)CH₂R where R is 4-pyridine or 1-imidazole. In some cases,phosphonate analogs may be employed as the analogs, e.g., where one ofR₂, R₃, R₄, R₉, R₁₀ or R₁₂ are not O, e.g., they are methyl etc. See,e.g., US patent application publication 2007-0072196, previouslyincorporated herein by reference in its entirety for all purposes.

The base moiety incorporated into the analog is generally selected fromany of the natural or non-natural nucleobases or nucleobase analogs,including, e.g., purine or pyrimidine bases that are routinely found innucleic acids and available nucleic acid analogs, including adenine,thymine, guanine, cytidine, uracil, and in some cases, inosine. Asnoted, the base optionally includes a label moiety. For convenience,nucleotides and nucleotide analogs are generally referred to based upontheir relative analogy to naturally occurring nucleotides. As such, ananalog that operates, functionally, like adenosine triphosphate, may begenerally referred to herein by the shorthand letter A. Likewise, thestandard abbreviations of T, G, C, U and I, may be used in referring toanalogs of naturally occurring nucleosides and nucleotides typicallyabbreviated in the same fashion. In some cases, a base may function in amore universal fashion, e.g., functioning like any of the purine basesin being able to hybridize with any pyrimidine base, or vice versa. Thebase moieties used in the present invention may include the conventionalbases described herein or they may include such bases substituted at oneor more side groups, or other fluorescent bases or base analogs, such as1,N6 ethenoadenosine or pyrrolo C, in which an additional ring structurerenders the B group neither a purine nor a pyrimidine. For example, incertain cases, it may be desirable to substitute one or more side groupsof the base moiety with a labeling group or a component of a labelinggroup, such as one of a donor or acceptor fluorophore, or other labelinggroup. Examples of labeled nucleobases and processes for labeling suchgroups are described in, e.g., U.S. Pat. Nos. 5,328,824 and 5,476,928,each of which is incorporated herein by reference in its entirety forall purposes.

In the analogs, the S group is optionally a sugar moiety that provides asuitable backbone for a synthesizing nucleic acid strand. For example,the sugar moiety is optionally selected from a D-ribosyl, 2′ or 3′D-deoxyribosyl, 2′,3′-D-dideoxyribosyl, 2′,3′-D-didehydrodideoxyribosyl,2′ or 3′ alkoxyribosyl, 2′ or 3′ aminoribosyl, 2′ or 3′ mercaptoribosyl,2′ or 3′ alkothioribosyl, acyclic, carbocyclic or other modified sugarmoieties. A variety of carbocyclic or acyclic moieties can beincorporated as the “S” group in place of a sugar moiety, including,e.g., those described in U.S. Patent Application Publication No.2003/0124576, which is incorporated herein by reference in its entiretyfor all purposes.

For most cases, the phosphorus containing chain in the analogs, e.g., atriphosphate in conventional NTPs, is preferably coupled to the 5′hydroxyl group, as in natural nucleoside triphosphates. However, in somecases, the phosphorus containing chain is linked to the S group by the3′ hydroxyl group.

L generally refers to a detectable labeling group that is coupled to theterminal phosphorus atom via the R₄ (or R₁₀ or R₁₂ etc.) group. Thelabeling groups employed in the analogs of the invention may compriseany of a variety of detectable labels. Detectable labels generallydenote a chemical moiety that provides a basis for detection of theanalog compound separate and apart from the same compound lacking such alabeling group. Examples of labels include, e.g., optical labels, e.g.,labels that impart a detectable optical property to the analog,electrochemical labels, e.g., labels that impart a detectable electricalor electrochemical property to the analog, and physical labels, e.g.,labels that impart a different physical or spatial property to theanalog, e.g., a mass tag or molecular volume tag. In some casesindividual labels or combinations may be used that impart more than oneof the aforementioned properties to the analogs of the invention.

Optionally, the labeling groups incorporated into the analogs compriseoptically detectable moieties, such as luminescent, chemiluminescent,fluorescent, fluorogenic, chromophoric and/or chromogenic moieties, withfluorescent and/or fluorogenic labels being preferred. A variety ofdifferent label moieties are readily employed in nucleotide analogs.Such groups include, e.g., fluorescein labels, rhodamine labels, cyaninelabels (i.e., Cy3, Cy5, and the like, generally available from theAmersham Biosciences division of GE Healthcare), and the Alexa family offluorescent dyes and other fluorescent and fluorogenic dyes availablefrom Molecular Probes/Invitrogen, Inc. and described in ‘The Handbook—AGuide to Fluorescent Probes and Labeling Technologies, Eleventh Edition’(2010) (available from Invitrogen, Inc./Molecular Probes). A variety ofother fluorescent and fluorogenic labels for use with nucleosidepolyphosphates, and which would be applicable to the nucleotide analogsincorporated by the polymerases of the present invention, are describedin, e.g., U.S. Patent Application Publication No. 2003/0124576,previously incorporated herein by reference in its entirety for allpurposes.

Thus, in one illustrative example, the analog can be a phosphate analog(e.g., an analog that has more than the typical number of phosphatesfound in nucleoside triphosphates) that includes, e.g., an Alexa dyelabel. For example, an Alexa488 dye can be labeled on a delta phosphateof a tetraphosphate analog (denoted, e.g., A488dC4P or A488dA4P, shownin FIG. 2, for the Alexa488 labeled tetraphosphate analogs of C and A,respectively), or an Alexa568 or Alexa633 dye can be used (e.g.,A568dC4P and A633dC4P, respectively, for labeled tetraphosphate analogsof C or A568dT6P for a labeled hexaphosphate analog of T), or anAlexa546 dye can be used (e.g., A546dG4P), or an Alexa594 dye can beused (e.g., A594dT4P). As additional examples, an Alexa555 dye (e.g.,A555dC6P or A555dA6P), an Alexa 647 dye (e.g., A647dG6P), an Alexa 568dye (e.g., A568dT6P), and/or an Alexa660 dye (e.g., A660dA6P orA660dC6P) can be used in, e.g., single molecule sequencing. Similarly,to facilitate color separation, a pair of fluorophores exhibiting FRET(fluorescence resonance energy transfer) can be labeled on a deltaphosphate of a tetraphosphate analog (denoted, e.g., FAM-amb-A532dG4P orFAM-amb-A594dT4P).

As noted above, an analog can include a linker that extends the distancebetween the nucleotide base and the label moiety, e.g., a fluorescentdye moiety. Exemplary linkers and analogs are described in U.S. Pat. No.7,968,702. Similarly, a protein or other moiety can be employed toprovide spacing and/or shielding between the base and the label, e.g.,as described in U.S. patent application 61/599,149, U.S. patentapplication Ser. No. 13/767,619 “Polymerase Enzyme Substrates withProtein Shield” filed Feb. 14, 2013, and U.S. patent application61/862,502 “Protected Fluorescent Reagent Compounds” filed Aug. 5, 2013.Suitable polymerase substrates optionally include two or more nucleosidepolyphosphates and/or two or more label moieties, e.g., as described inU.S. patent application 61/599,149 “Polymerase Enzyme Substrates withProtein Shield,” U.S. patent application Ser. No. 13/767,619 “PolymeraseEnzyme Substrates with Protein Shield,” U.S. patent application61/862,502 “Protected Fluorescent Reagent Compounds,” and US patentapplication publication 2009-0208957 Alternate Labeling Strategies forSingle Molecule Sequencing.

Additional details regarding labels, analogs, and methods of making suchanalogs can be found in US patent application publication 2007-0072196,WO 2007/041342 Labeled Nucleotide Analogs and Uses Therefor, WO2009/114182 Labeled Reactants and Their Uses, US patent applicationpublication 2009-0208957 Alternate Labelling Strategies for SingleMolecule Sequencing, U.S. patent application Ser. No. 13/218,412Functionalized Cyanine Dyes, U.S. patent application Ser. No. 13/218,395Functionalized Cyanine Dyes, U.S. patent application Ser. No. 13/218,428Cyanine Dyes, U.S. patent application Ser. No. 13/218,382 Scaffold-BasedPolymerase Enzyme Substrates, US patent application publication2010-0167299 Phospholink Nucleotides for Sequencing Applications, USpatent application publication 2010-0152424 Modular NucleotideCompositions and Uses Therefor, U.S. patent application 61/599,149Polymerase Enzyme Substrates with Protein Shield, U.S. patentapplication Ser. No. 13/767,619 “Polymerase Enzyme Substrates withProtein Shield,” and U.S. patent application 61/862,502 “ProtectedFluorescent Reagent Compounds,” each of which is incorporated herein byreference in its entirety for all purposes.

Applications for Enhanced Nucleic Acid Amplification and Sequencing

Polymerases of the invention, e.g., modified recombinant polymerases,are optionally used in combination with nucleotides and/or nucleotideanalogs and nucleic acid templates (e.g., DNA, RNA, or hybrids, analogs,derivatives, or mimetics thereof) to copy the template nucleic acid.That is, a mixture of the polymerase, nucleotides/analogs, andoptionally other appropriate reagents, the template and a replicationinitiating moiety (e.g., primer) is reacted such that the polymerasesynthesizes nucleic acid (e.g., extends the primer) in atemplate-dependent manner. The replication initiating moiety can be astandard oligonucleotide primer, or, alternatively, a component of thetemplate, e.g., the template can be a self-priming single stranded DNA,a nicked double stranded DNA, or the like. Similarly, a terminal proteincan serve as an initiating moiety. At least one nucleotide analog can beincorporated into the DNA. The template DNA can be a linear or circularDNA, and in certain applications, is desirably a circular template(e.g., for rolling circle replication or for sequencing of circulartemplates). Optionally, the composition can be present in an automatedDNA replication and/or sequencing system.

Incorporation of labeled nucleotide analogs by the polymerases of theinvention is particularly useful in a variety of different nucleic acidanalyses, including real-time monitoring of DNA polymerization. Thelabel can itself be incorporated, or more preferably, can be releasedduring incorporation of the analog. For example, analog incorporationcan be monitored in real time by monitoring label release duringincorporation of the analog by the polymerase. The portion of the analogthat is incorporated can be the same as a natural nucleotide, or caninclude features of the analog that differ from a natural nucleotide.

In general, label incorporation or release can be used to indicate thepresence and composition of a growing nucleic acid strand, e.g.,providing evidence of template replication/amplification and/or sequenceof the template. Signaling from the incorporation can be the result ofdetecting labeling groups that are liberated from the incorporatedanalog, e.g., in a solid phase assay, or can arise upon theincorporation reaction. For example, in the case of FRET labels where abound label is quenched and a free label is not, release of a labelgroup from the incorporated analog can give rise to a fluorescentsignal. Alternatively, the enzyme may be labeled with one member of aFRET pair proximal to the active site, and incorporation of an analogbearing the other member will allow energy transfer upon incorporation.The use of enzyme bound FRET components in nucleic acid sequencingapplications is described, e.g., in U.S. Patent Application PublicationNo. 2003/0044781, incorporated herein by reference.

In one example reaction of interest, a polymerase reaction can beisolated within an extremely small observation volume that effectivelyresults in observation of individual polymerase molecules. As a result,the incorporation event provides observation of an incorporatingnucleotide analog that is readily distinguishable from non-incorporatednucleotide analogs. In a preferred aspect, such small observationvolumes are provided by immobilizing the polymerase enzyme within anoptical confinement, such as a Zero Mode Waveguide (ZMW). For adescription of ZMWs and their application in single molecule analyses,and particularly nucleic acid sequencing, see, e.g., U.S. PatentApplication Publication No. 2003/0044781 and U.S. Pat. No. 6,917,726,each of which is incorporated herein by reference in its entirety forall purposes. See also Levene et al. (2003) “Zero-mode waveguides forsingle-molecule analysis at high concentrations” Science 299:682-686,Eid et al. (2009) “Real-time DNA sequencing from single polymerasemolecules” Science 323:133-138, and U.S. Pat. Nos. 7,056,676, 7,056,661,7,052,847, and 7,033,764, the full disclosures of which are incorporatedherein by reference in their entirety for all purposes.

In general, a polymerase enzyme is complexed with the template strand inthe presence of one or more nucleotides and/or one or more nucleotideanalogs. For example, in certain embodiments, labeled analogs arepresent representing analogous compounds to each of the four naturalnucleotides, A, T, G and C, e.g., in separate polymerase reactions, asin classical Sanger sequencing, or multiplexed together, e.g., in asingle reaction, as in multiplexed sequencing approaches. When aparticular base in the template strand is encountered by the polymeraseduring the polymerization reaction, it complexes with an availableanalog that is complementary to such nucleotide, and incorporates thatanalog into the nascent and growing nucleic acid strand. In one aspect,incorporation can result in a label being released, e.g., inpolyphosphate analogs, cleaving between the a and 13 phosphorus atoms inthe analog, and consequently releasing the labeling group (or a portionthereof). The incorporation event is detected, either by virtue of alonger presence of the analog and, thus, the label, in the complex, orby virtue of release of the label group into the surrounding medium.Where different labeling groups are used for each of the types ofanalogs, e.g., A, T, G or C, identification of a label of anincorporated analog allows identification of that analog andconsequently, determination of the complementary nucleotide in thetemplate strand being processed at that time. Sequential reaction andmonitoring permits real-time monitoring of the polymerization reactionand determination of the sequence of the template nucleic acid. As notedabove, in particularly preferred aspects, the polymerase enzyme/templatecomplex is provided immobilized within an optical confinement thatpermits observation of an individual complex, e.g., a zero modewaveguide. For additional information on single molecule sequencingmonitoring incorporation of phosphate-labeled analogs in real time, see,e.g., Eid et al. (2009) “Real-time DNA sequencing from single polymerasemolecules” Science 323:133-138.

In a first exemplary technique, as schematically illustrated in FIG. 3A,a nucleic acid synthesis complex, including a polymerase enzyme 202, atemplate sequence 204 and a complementary primer sequence 206, isprovided immobilized within an observation region 200 that permitsillumination (as shown by hv) and observation of a small volume thatincludes the complex without excessive illumination of the surroundingvolume (as illustrated by dashed line 208). By illuminating andobserving only the volume immediately surrounding the complex, one canreadily identify fluorescently labeled nucleotides that becomeincorporated during that synthesis, as such nucleotides are retainedwithin that observation volume by the polymerase for longer periods thanthose nucleotides that are simply randomly diffusing into and out ofthat volume.

In particular, as shown in FIG. 3B, when a nucleotide, e.g., A, isincorporated into DNA by the polymerase, it is retained within theobservation volume for a prolonged period of time, and upon continuedillumination yields a prolonged fluorescent signal (shown by peak 210).By comparison, randomly diffusing and not incorporated nucleotidesremain within the observation volume for much shorter periods of time,and thus produce only transient signals (such as peak 212), many ofwhich go undetected due to their extremely short duration.

In particularly preferred exemplary systems, the confined illuminationvolume is provided through the use of arrays of optically confinedapertures termed zero mode waveguides (ZMWs), e.g., as shown by confinedreaction region 200 (see, e.g., U.S. Pat. No. 6,917,726, which isincorporated herein by reference in its entirety for all purposes). Forsequencing applications, the DNA polymerase is typically providedimmobilized upon the bottom of the ZMW, although another component ofthe complex (e.g., a primer or template) is optionally immobilized onthe bottom of the ZMW to localize the complex. See, e.g., Korlach et al.(2008) PNAS U.S.A. 105(4):1176-1181 and US patent applicationpublication 2008-0032301, each of which is incorporated herein byreference in its entirety for all purposes.

In operation, the fluorescently labeled nucleotides (shown as A, C, Gand T) bear one or more fluorescent dye groups on a terminal phosphatemoiety that is cleaved from the nucleotide upon incorporation. As aresult, synthesized nucleic acids do not bear the build-up offluorescent labels, as the labeled polyphosphate groups diffuse awayfrom the complex following incorporation of the associated nucleotide,nor do such labels interfere with the incorporation event. See, e.g.,Korlach et al. (2008) Nucleosides, Nucleotides and Nucleic Acids27:1072-1083.

A fluorescence time trace for a ZMW, showing pulses (peaks) representingincorporation of different nucleotide analogs, is presented in FIG. 4. Apulse width and interpulse distance are illustrated on the trace. Theinset schematically illustrates the catalytic cycle forpolymerase-mediated nucleic acid primer extension according to theexemplary reaction scheme described in US patent application publication2012-0034602; the box indicates the portion of the catalytic cycle thatcorresponds to the pulse when sequencing is performed withphosphate-labeled nucleotide analogs. The remainder of the cyclecorresponds to the interpulse distance.

In a second exemplary technique, the immobilized complex and thenucleotides to be incorporated are each provided with interactivelabeling components. Upon incorporation, the nucleotide borne labelingcomponent is brought into sufficient proximity to the complex borne (orcomplex proximal) labeling component, such that these components producea characteristic signal event. For example, the polymerase may beprovided with a fluorophore that provides fluorescent resonant energytransfer (FRET) to appropriate acceptor fluorophores. These acceptorfluorophores are provided upon the nucleotide to be incorporated, whereeach type of nucleotide bears a different acceptor fluorophore, e.g.,that provides a different fluorescent signal. Upon incorporation, thedonor and acceptor are brought close enough together to generate energytransfer signal. By providing different acceptor labels on the differenttypes of nucleotides, one obtains a characteristic FRET-basedfluorescent signal for the incorporation of each type of nucleotide, asthe incorporation is occurring.

In a related aspect, a nucleotide analog may include two interactingfluorophores that operate as a donor/quencher pair, where one member ispresent on the nucleobase or other retained portion of the nucleotide,while the other member is present on a phosphate group or other portionof the nucleotide that is released upon incorporation, e.g., a terminalphosphate group. Prior to incorporation, the donor and quencher aresufficiently proximal on the same analog as to provide characteristicsignal quenching. Upon incorporation and cleavage of the terminalphosphate groups, e.g., bearing a donor fluorophore, the quenching isremoved and the resulting characteristic fluorescent signal of the donoris observable.

In exploiting the foregoing processes, where the incorporation reactionoccurs too rapidly, it may result in the incorporation event not beingdetected, i.e., the event speed exceeds the detection speed of themonitoring system. The missed detection of incorporated nucleotides canlead to an increased rate of errors in sequence determination, asomissions in the real sequence. In order to mitigate the potential formissed pulses due to short reaction or product release times, in oneaspect, the current invention can result in increased reaction and/orproduct release times during incorporation cycles. Similarly, very shortinterpulse distances can occasionally cause pulse merging. An advantageof employing polymerases with reduced reaction rates, e.g., polymerasesexhibiting decreased rates and/or two slow-step kinetics as described inUS patent application publications 2009-0286245 and 2010-0112645, is anincreased frequency of longer, detectable, binding events. Thisadvantage may also be seen as an increased ratio of longer, detectablepulses to shorter, non-detectable pulses, where the pulses representbinding events.

In addition to their use in sequencing, the polymerases of the inventionare also useful in a variety of other genotyping analyses, e.g., SNPgenotyping using single base extension methods, real time monitoring ofamplification, e.g., RT-PCR methods, and the like. The polymerases ofthe invention are also useful in amplifying nucleic acids, e.g., DNAs orRNAs, including, for example, in applications such as whole genomeamplification. For example, polymerases of the invention that showincreased thermostability or resistance to organic solvents (e.g.,DMSO), or that otherwise exhibit an improved ability to read throughdamaged, modified, or other “difficult” stretches of nucleic acidtemplate, can be suitably employed in whole genome amplification. Forreview of whole genome amplification, see, e.g., Silander and Saarela(2008) “Whole Genome Amplification with Phi29 DNA Polymerase to EnableGenetic or Genomic Analysis of Samples of Low DNA Yield” Methods inMolecular Biology 439:1-18 and Pinard et al. (2006) “Assessment of wholegenome amplification-induced bias through high-throughput, massivelyparallel whole genome sequencing” BMC Genomics 7:216. Further detailsregarding sequencing and nucleic acid amplification can be found, e.g.,in Sambrook, Ausubel, and Innis, all infra.

Recombinant Polymerases with Increased Stability and Readlength

The compositions of the invention comprise a modified recombinant DNApolymerase which exhibits one or more altered properties desirable insingle molecule sequencing applications or other applications involvingnucleic acid synthesis. An exemplary property of certain polymerases ofthe invention is increased stability (e.g., thermostability, e.g., of apolymerase-DNA substrate binary complex) relative to a wild-type orparental polymerase. Other exemplary properties include increasedreadlength, enhanced utility with large nucleotide analogs, alteredkinetic behavior (e.g., demonstration of slow catalytic steps),exonuclease deficiency, increased closed complex stability, altered(e.g., reduced) branching fraction, altered cofactor selectivity,increased yield, increased cosolvent resistance, increasedphototolerance, increased accuracy, and increased speed.

As will be understood, a polymerase of the invention can display one ofthe aforementioned properties alone or can display two or more of theproperties in combination. Moreover, it will be understood that while apolymerase or group of polymerases may be described with respect to aparticular property, the polymerase(s) may possess additional modifiedproperties not mentioned in every instance for ease of discussion. Itwill also be understood that particular properties are observed undercertain conditions. For example, a stability-improving mutation can,e.g., confer increased stability on the polymerase-DNA substrate binarycomplex (as compared to such a complex containing a parental polymeraselacking the mutation) when observed in a thermal inactivation assay orit can confer increased readlength when observed in a single moleculesequencing reaction where the lifetime of the parental polymerase-DNAsubstrate complex (and therefore readlength) is limited by itsstability. A single mutation (e.g., a single amino acid substitution,deletion, insertion, or the like) may give rise to the one or morealtered properties, or the one or more properties may result from two ormore mutations which act in concert to confer the desired activity. Therecombinant polymerases, mutations, and altered properties exhibited bythe recombinant polymerases are set forth in greater detail below.

As noted, various combinations of the individual mutations describedherein can be introduced into recombinant polymerases to confer avariety of advantageous properties on the polymerases. However,introducing additional mutations into a polymerase can have deleteriouseffects on its thermostability. Without limitation to any particularmechanism, thermostability of the free polymerase (uncomplexed with DNAsubstrate and/or nucleotide analog) is in many cases generallycorrelated with yield when the polymerase is purified. Mutations thatincrease protein thermostability are thus desirable, not only becausesuch mutations often also increase protein yield, but also becauseincreased thermostability can result in longer lifetime of thepolymerase, e.g., during storage, under assay conditions used in singlemolecule sequencing or nucleic amplification at elevated temperatures,or the like. (See, e.g., US patent application publication2012-0034602.)

Protein thermostability can be assayed by any of a variety of techniquesknown in the art. For example, polymerase thermostability can beassessed basically as described in Vedadi et al. (2006) Proc Natl AcadSci 103:15835-15840. Purified polymerase is incubated with theflorescent dye SYPRO® orange, which binds more strongly to partiallyunfolded protein than to folded or unfolded protein. Fluorescence ismonitored as the temperature is increased. The unfolding temperature isdetermined as the temperature of the midpoint between the initialminimum and maximum in florescent intensity. A recombinant polymerasewith increased thermostability thus has a higher unfolding temperature,while a polymerase with decreased thermostability has a lower unfoldingtemperature.

Positions relative to a wild-type Φ29 polymerase that can be mutated toincrease thermostability of the free polymerase include, e.g., Y224,V250, T368, E508, E515, and/or F526. Exemplary substitutions that canenhance polymerase stability include, e.g., Y224K, V250I, T368F, T368Y,E508R, E515Q, E515K, and F526L.

Thermostability can also be assessed by measuring activity afterincubation of the polymerase at different temperatures, optionally inthe presence of a substrate and nucleotides or nucleotide analogs.Without limitation to any particular mechanism, stability of apolymerase-DNA substrate-nucleotide ternary complex (e.g., a complexincluding a polymerase, a primer and template, and a nucleotide ornucleotide analog) and/or stability of a polymerase-DNA substrate binarycomplex (e.g., including a polymerase and a primer and template) is inmany cases generally correlated with readlength, e.g., readlengthobserved in single molecule sequencing.

An exemplary thermal inactivation assay in which the stability of aternary complex including the polymerase, a gapped duplex DNA substrate,and a cognate nucleotide or nucleotide analog is assessed is describedin US patent application publication 2012-0034602. Positions relative toa wild-type Φ29 polymerase that can be mutated to increasethermostability of the ternary complex include, e.g., V250, L253, T368,A484, E515, and F526. Exemplary substitutions that can enhance ternarycomplex stability include, e.g., V250I, L253H in combination with A437G,T368V, A484E, E515Q, E515K, and F526L. It is worth noting that stabilityof the ternary complex can also be influenced by the identity of othercomponents of the complex, particularly the nucleotide or analog (see USpatent application publication 2012-0034602).

Another exemplary thermal inactivation assay is schematicallyillustrated in FIG. 5A. A complex including the polymerase, a gappedduplex DNA substrate bearing a fluorophore and a quencher, and a cognatenucleotide triphosphate or nucleotide analog (e.g., dATP or ahexaphosphate or other analog thereof) is assembled in the presence ofSr⁺⁺. Mg⁺⁺ is then added to permit incorporation of the cognatenucleotide or analog. Since the other three nucleotides are not present,a binary complex including the polymerase and the DNA substrate(extended by two bases, in the example shown in FIG. 5A) results. Anexcess of heparin is also added, such that any polymerase thatdissociates from the DNA substrate binds to the heparin. Samples of thebinary complex are exposed, e.g., to temperatures between 30° C. and 50°C. for 30 minutes. The remaining nucleotides (e.g., the other threedNTPs) are then added; polymerase which has remained active and bound tothe substrate displaces the oligonucleotide bearing the quencher,producing a fluorescent signal.

Thermal inactivation profiles for a series of Φ29 recombinantpolymerases are shown in FIG. 5B. As seen in FIG. 5B, addition of aD570E substitution to Φ29 polymerases carrying other substitutions canincrease stability of the binary complex.

Restoring the binary stability of polymerases destabilized by theintroduction of other mutations can enhance readlength. As shown in FIG.5C, addition of a D570E substitution to a variety of recombinant Φ29polymerases increases both their binary stability (indicated by thebinary IT50 value shown over the graph, where the IT50 is the midpointof the observed thermal inactivation profile) and their readlength. Thecontrol polymerase includes K131E, Y148I, Y224K, E239G, V250I, L253A,E375Y, A437G, A484E, D510K, K512Y, and E515Q substitutions. The otherpolymerases include K131E, Y148I, Y224K, E239G, V250I, L253A, E375Y,A437G, A484E, D510K, and K512Y substitutions in addition to thesubstitution noted on the graph, with or without a D570E substitution.Each of the polymerases also includes a C-terminal His10 tag andbiotinylation site (BtagV7).

Positions relative to a wild-type Φ29 polymerase that can be mutated toenhance binary stability and/or readlength include, e.g., Q99, Y148,K131, and D570. Exemplary substitutions that can enhance binarystability include, e.g., Q99I, Y148I, K131E, D570E, D570S, D570T, D570M,D570V, D570W, D570G, and D570C. Such mutations are optionally employedin combination with each other to further enhance stability; forexample, the effects of D570E and Y148I on binary stability can beapproximately additive.

Mutations that increase the thermostability of the free polymerase donot always increase binary complex and/or ternary complex stability, andvice versa; for example, mutations that increase binary complexstability do not always increase free polymerase and ternary complexstability. For example, a Y224K substitution can increasethermostability of the free polymerase but does not appear tosignificantly increase binary complex stability, and an F526Lsubstitution can increase both thermostability of the free polymeraseand stability of the ternary complex but does not appear tosignificantly increase stability of the binary complex.

The assays described herein (e.g., for binary complex, ternary complex,and free polymerase stability) are optionally employed in screeningmutant polymerases to identify recombinant polymerases of particularinterest. For example, a parental polymerase can be mutated at one ormore positions, then its binary complex, ternary complex, or freepolymerase stability can be assessed. One or more additional propertiescan be assessed for any mutant polymerase showing altered (e.g.,increased) binary complex, ternary complex, or free polymerasestability, e.g., readlength and/or yield.

As will be appreciated, recombinant polymerases that exhibit increasedstability and/or readlength can also include additional mutations (e.g.,amino acid substitutions, deletions, insertions, exogenous features,e.g., at the N- and/or C-terminus, and/or the like) which confer one ormore additional desirable properties as described herein or known in theart, e.g., enhanced utility with large nucleotide analogs, reduced oreliminated exonuclease activity, convenient surface immobilization,improved sensitivity to base modifications, increased closed complexstability, reduced or increased branching, selectivity for particularmetal cofactors, increased yield, increased accuracy, increased speed,and/or increased phototolerance.

Recombinant Polymerases for Incorporation of Large Analogs

Detection of optical labels in an enzymatic reaction generally entailsdirecting excitation radiation at the reaction mixture to excite alabeling group present in the mixture, which is then separatelydetectable. However, prolonged exposure of chemical and biochemicalreactants to radiation (e.g., light) energy during the excitation anddetection of optical labels can damage components of the reactionmixture, e.g., enzymes, proteins, substrates, or the like. For example,it has been observed that, in template-directed synthesis of nucleicacids from fluorescently labeled nucleotides or nucleotide analogs,sustained exposure of the DNA polymerase to excitation radiation used inthe detection of the relevant label (e.g., fluorophore) reduces theenzyme's processivity and polymerase activity. Although illuminatedreactions typically proceed under conditions where the reactants (e.g.,enzyme molecules, etc.) are present in excess such that any adverseeffects of photodamage on any single enzyme molecule in the reaction mixdo not, in general, affect operation of the assay, an increasing numberof analyses that entail the use of optical labels are performed withreactants at very low concentrations. For example, polymerases can beused to synthesize DNAs from fluorescently labeled nucleotide analogs inmicrofluidic or nanofluidic reaction vessels or channels or in opticallyconfined reaction volumes, e.g., in a zero-mode waveguide (ZMW) or ZMWarray as described above. Analysis of small, single-analyte reactionvolumes is becoming increasingly important in high-throughputapplications, e.g., in DNA sequencing. However, in such reactant-limitedanalyses, any degradation of a critical reagent such as an enzymemolecule due to photodamage can dramatically interfere with theanalysis.

Without limitation to any particular mechanism, observation ofpolymerase performance in single molecule sequencing reactions usinglabeled nucleotide analogs has revealed that, in many instances,photodamage involves collision of the dye moiety of an analog with thepolymerase followed by cros slink formation between the dye and thepolymerase. One approach to increasing polymerase phototolerance thusinvolves reducing the frequency of such collisions. For example, asdescribed in U.S. patent application Ser. No. 13/756,113 “RecombinantPolymerases With Increased Phototolerance,” since the nucleotide analogis negatively charged, the frequency of collisions between thepolymerase and the label can be reduced by introducing negative chargesto and/or removing positive charges from the surface of the polymerasethat is within reach of the dye moiety. As another example, againwithout limitation to any particular mechanism, photodamage to thepolymerase can be reduced by employing one or more nucleotide analogsdesigned to reduce photodamage, e.g., by increasing the effectivedistance the dye moiety is maintained from the surface of the polymeraseor by otherwise shielding the polymerase from collision with the dye.

Exemplary analogs designed to reduce photodamage are described in USpatent application publication 2013/0316912 of application Ser. No.13/767,619 “Polymerase Enzyme Substrates with Protein Shield” by KeithBjornson et al. filed Feb. 14, 2013 (incorporated herein by reference inits entirety for all purposes) and include protein-shielded analogs thatcomprise an avidin protein (e.g., a tetrameric biotin-binding protein,avidin, streptavidin, neutravidin, tamavidin, or the like), ubiquitin,or another polypeptide moiety (e.g., a polypeptide comprising at least60 amino acids) separating the dye and the nucleotide components of theanalog (e.g., attached to and positioned between the phosphate portionof a nucleoside polyphosphate and the fluorescent dye). An exemplaryprotein shield analog comprises a protein, e.g., comprising at least 60amino acids (e.g., 60 to 1,000 amino acids, e.g., 80 to 600 aminoacids), a nucleotide component comprising at least one nucleosidepolyphosphate attached through its phosphate portion to a first positionon the protein, and a dye component comprising at least one fluorescentdye moiety attached to a second position on the protein. The first andsecond attachment points are spaced apart by a distance such that when anucleoside phosphate attached to the protein component of the analog isin the active site of the polymerase enzyme, a fluorescent dye moietyattached to the protein is shielded by the protein from coming intocontact with the polymerase. The analog optionally includes two or morenucleoside phosphates and/or two or more fluorescent dye moieties. Eachnucleoside polyphosphate optionally includes three or more phosphategroups, e.g., 4-7 or more phosphates. The nucleotide and/or dyecomponents can be covalently or noncovalently attached to the proteincomponent of the analog. For example, the protein component of theanalog can be a biotin-binding protein, to which biotinylated nucleosidepolyphosphate and/or fluorescent dye moieties are bound. In one class ofembodiments, the analog comprises an avidin protein having foursubunits, each of which includes one biotin binding site; one or twonucleotide components each comprising one or more phospholinkednucleotide moieties; and one or two dye components each comprising oneor more dye moieties. Each nucleotide and dye component is bound to theavidin protein through a biotin moiety attached to a binding site on theavidin protein. Preferably, at least one of the nucleotide or dyecomponents comprises a bis-biotin moiety bound to two of the biotinbinding sites on the avidin protein. Optionally, the avidin protein isstreptavidin or a homolog or variant thereof. See US patent applicationpublication 2013/0316912 of application Ser. No. 13/767,619 foradditional details on and examples of protein shield analogs.

Additional exemplary analogs designed to reduce photodamage aredescribed in U.S. patent application 61/862,502 “Protected FluorescentReagent Compounds” by Lubomir Sebo et al. filed Aug. 5, 2013(incorporated herein by reference in its entirety for all purposes) andinclude shielded analogs that comprise a multivalent central coreelement being or comprising at least one fluorescent dye. Surroundingthe core are multiple intermediate chemical groups, at least one ofwhich comprises a shield element, and multiple terminal chemical groups,at least one of which comprises a nucleotide. The analog optionallyincludes 2-24 nucleotides, or even more than 24 nucleotides. Exemplaryshield elements include neutral side chains (e.g., polyethylene glycol)and negative side chains (e.g., sulfonic acid). See U.S. patentapplication 61/862,502 for additional details on and examples ofshielded analogs. Suitable analogs also include those described in U.S.Pat. No. 7,968,702 (incorporated herein by reference in its entirety forall purposes), in which the label is maintained at least 2 nm from thepolymerase.

The analog optionally has a molecular weight (i.e., molecular mass) ofat least 10,000, for example, a molecular weight of at least 20,000, atleast 30,000, at least 40,000, at least 50,000, or even at least 60,000,at least 100,000, at least 200,000, or at least 300,000 Da.

As will be evident, such analogs are considerably larger than thosetypically employed in nucleic acid synthesis and sequencing, and theiruse can therefore be facilitated by modifying the polymerase to enhancetheir incorporation and/or to compensate for undesirable effects of theanalogs' size (e.g., altered pulse characteristics or an increasedfrequency of cognate extra errors in which a cognate analog binds to thetemplate but dissociates before being incorporated, producing a spuriouspulse and thus an insertion error).

The polymerase optionally includes one or more substitutions to enhanceperformance of the polymerase with such analogs, e.g., in singlemolecule sequencing. For example, where use of large analogs undesirablynarrows pulse width, one or more substitutions that increase pulse width(e.g., P558A, P558F, A256S and/or S487A) can be included in thepolymerase. Where use of large analogs undesirably increases interpulsedistance, the polymerase can include one or more substitutions thatdecrease interpulse distance (e.g., V141K, L142K, E466K, D476H, E508R,L513K, and/or D523R). Pausing when large analogs are employed can bereduced by inclusion in the polymerase of one or more substitutions suchas, e.g., Q99P, R306Q, R308L, K311E, and/or T441I. A polymerase for usewith large analogs can also include one or more substitutions to improvestability as described above (e.g., binary complex stability, e.g.,D570E or D570M, and/or free polymerase or ternary complex stability,e.g., F526L), accuracy (e.g., Q99Y, K536T, K536E, A134S and/or I524S,optionally in combination with F526L to compensate for undesirableeffects of I524S on stability or yield), and/or alter another polymeraseproperty as described herein (e.g., Y224K, E239G, L253A, E375Y, A437G,A437N, A484E, D510K, K512Y, E515Q, D570S, and/or T571V). Exemplarycombinations of substitutions useful in polymerases employed in singlemolecule sequencing with large analogs are provided hereinbelow. See,e.g., the section entitled “Combining Mutations” and Tables 7-10hereinbelow, as well as FIG. 9; certain selected combinations includedin Tables 3-6 and FIG. 7 are also suitable for use with large analogs.

Design and Characterization of Recombinant Polymerases

In addition to methods of using the polymerases and other compositionsherein, the present invention also includes methods of making thepolymerases. (Polymerases made by the methods are also a feature of theinvention, and it will be evident that, although various designstrategies are detailed herein, no limitation of the resultingpolymerases to any particular mechanism is thereby intended.) Asdescribed, methods of making a recombinant DNA polymerase can includestructurally modeling a parental polymerase, e.g., using any availablecrystal structure and molecular modeling software or system. Based onthe modeling, one or more amino acid residue positions in the polymeraseare identified as targets for mutation. For example, one or more featureaffecting stability (e.g., thermostability of the free polymerase,binary complex stability, and/or ternary complex stability),phototolerance, closed complex stability, nucleotide access to orremoval from the active site (and, thereby, branching), binding of a DNAor nucleotide analog, product binding, etc. is identified. Theseresidues can be, e.g., in the active site or a binding pocket or in adomain such as the exonuclease, TPR2 or thumb domain (or interfacebetween domains) or proximal to such domains. The DNA polymerase ismutated to include different residues at such positions (e.g., anotherone of the nineteen other commonly occurring natural amino acids or anon-natural amino acid, e.g., a nonpolar and/or aliphatic residue, apolar uncharged residue, an aromatic residue, a positively chargedresidue, or a negatively charged residue), and then screened for anactivity of interest (e.g., stability (e.g., thermostability of the freepolymerase, binary complex stability, and/or ternary complex stability),readlength, sensitivity to base modification, analog incorporation,phototolerance, processivity, k_(off), K_(d), branching fraction,decreased rate constant, balanced rate constants, accuracy, speed,yield, cofactor selectivity, cosolvent resistance, etc.). It will beevident that catalytic and/or highly conserved residues are typically(but not necessarily) less preferred targets for mutation.

Further, as noted above, a polymerase of the invention (e.g., a Φ29-typeDNA polymerase that includes E375, K512, L253, and/or A484 mutations)can be further modified to enhance the properties of the polymerase. Forexample, a polymerase comprising a combination of the above mutationscan be mutated at one or more additional sites to enhance a propertyalready possessed by the polymerase or to confer a new property notprovided by the existing mutations. Details correlating polymerasestructure with desirable functionalities that can be added topolymerases of the invention are provided herein. Also provide below arevarious approaches for modifying/mutating polymerases of the invention,determining kinetic parameters or other properties of the modifiedpolymerases, screening modified polymerases, and adding exogenousfeatures to the N- and/or C-terminal regions of the polymerases.

Structure-Based Design of Recombinant Polymerases

Structural data for a polymerase can be used to conveniently identifyamino acid residues as candidates for mutagenesis to create recombinantpolymerases, for example, having modified active site regions and/ormodified domain interfaces to increase polymerase stability, improvecomplex stability, increase readlength, increase phototolerance, reducereaction rates, reduce branching, reduce exonuclease activity, altercofactor selectivity, improve yield, or confer other desirableproperties. For example, analysis of the three-dimensional structure ofa polymerase such as Φ29 can identify residues that are in the activepolymerization site of the enzyme, residues that form part of thenucleotide analog binding pocket, and/or amino acids at an interfacebetween domains.

The three-dimensional structures of a large number of DNA polymeraseshave been determined by x-ray crystallography and nuclear magneticresonance (NMR) spectroscopy, including the structures of polymeraseswith bound templates, nucleotides, and/or nucleotide analogs. Many suchstructures are freely available for download from the Protein Data Bank,at (www(dot)rcsb(dot)org/pdb. Structures, along with domain and homologyinformation, are also freely available for search and download from theNational Center for Biotechnology Information's Molecular ModelingDataBase, atwww(dot)ncbi(dot)nlm(dot)nih(dot)gov/Structure/MMDB/mmdb(dot)shtml. Thestructures of Φ29 polymerase, Φ29 polymerase complexed with terminalprotein, and Φ29 polymerase complexed with primer-template DNA in thepresence and absence of a nucleoside triphosphate are available; seeKamtekar et al. (2004) “Insights into strand displacement andprocessivity from the crystal structure of the protein-primed DNApolymerase of bacteriophage Φ29” Mol. Cell 16(4): 609-618), Kamtekar etal. (2006) “The phi29 DNA polymerase:protein-primer structure suggests amodel for the initiation to elongation transition” EMBO J.25(6):1335-43, and Berman et al. (2007) “Structures of phi29 DNApolymerase complexed with substrate: The mechanism of translocation inB-family polymerases” EMBO J. 26:3494-3505, respectively. The structuresof additional polymerases or complexes can be modeled, for example,based on homology of the polymerases with polymerases whose structureshave already been determined. Alternatively, the structure of a givenpolymerase (e.g., a wild-type or modified polymerase), optionallycomplexed with a DNA (e.g., template and/or primer) and/or nucleotideanalog, or the like, can be determined.

Techniques for crystal structure determination are well known. See, forexample, McPherson (1999) Crystallization of Biological MacromoleculesCold Spring Harbor Laboratory; Bergfors (1999) Protein CrystallizationInternational University Line; Mullin (1993) CrystallizationButterwoth-Heinemann; Stout and Jensen (1989) X-ray structuredetermination: a practical guide, 2nd Edition Wiley Publishers, NewYork; Ladd and Palmer (1993) Structure determination by X-raycrystallography, 3rd Edition Plenum Press, New York; Blundell andJohnson (1976) Protein Crystallography Academic Press, New York; Gluskerand Trueblood (1985) Crystal structure analysis: A primer, 2nd Ed.Oxford University Press, New York; International Tables forCrystallography, Vol. F. Crystallography of Biological Macromolecules;McPherson (2002) Introduction to Macromolecular CrystallographyWiley-Liss; McRee and David (1999) Practical Protein Crystallography,Second Edition Academic Press; Drenth (1999) Principles of Protein X-RayCrystallography (Springer Advanced Texts in Chemistry) Springer-Verlag;Fanchon and Hendrickson (1991) Chapter 15 of Crystallographic Computing,Volume 5 IUCr/Oxford University Press; Murthy (1996) Chapter 5 ofCrystallographic Methods and Protocols Humana Press; Dauter et al.(2000) “Novel approach to phasing proteins: derivatization by shortcryo-soaking with halides” Acta Cryst. D56:232-237; Dauter (2002) “Newapproaches to high-throughput phasing” Curr. Opin. Structural Biol.12:674-678; Chen et al. (1991) “Crystal structure of a bovineneurophysin-II dipeptide complex at 2.8 Å determined from thesingle-wavelength anomalous scattering signal of an incorporated iodineatom” Proc. Natl Acad. Sci. USA, 88:4240-4244; and Gavira et al. (2002)“Ab initio crystallographic structure determination of insulin fromprotein to electron density without crystal handling” Acta Cryst.D58:1147-1154.

In addition, a variety of programs to facilitate data collection, phasedetermination, model building and refinement, and the like are publiclyavailable. Examples include, but are not limited to, the HKL2000 package(Otwinowski and Minor (1997) “Processing of X-ray Diffraction DataCollected in Oscillation Mode” Methods in Enzymology 276:307-326), theCCP4 package (Collaborative Computational Project (1994) “The CCP4suite: programs for protein crystallography” Acta Crystallogr D50:760-763), SOLVE and RESOLVE (Terwilliger and Berendzen (1999) ActaCrystallogr D 55 (Pt 4):849-861), SHELXS and SHELXD (Schneider andSheldrick (2002) “Substructure solution with SHELXD” Acta Crystallogr DBiol Crystallogr 58:1772-1779), Refmac5 (Murshudov et al. (1997)“Refinement of Macromolecular Structures by the Maximum-LikelihoodMethod” Acta Crystallogr D 53:240-255), PRODRG (van Aalten et al. (1996)“PRODRG, a program for generating molecular topologies and uniquemolecular descriptors from coordinates of small molecules” J ComputAided Mol Des 10:255-262), and Coot (Elmsley et al. (2010) “Features andDevelopment of Coot” Acta Cryst D 66:486-501.

Techniques for structure determination by NMR spectroscopy are similarlywell described in the literature. See, e.g., Cavanagh et al. (1995)Protein NMR Spectroscopy: Principles and Practice, Academic Press;Levitt (2001) Spin Dynamics: Basics of Nuclear Magnetic Resonance, JohnWiley & Sons; Evans (1995) Biomolecular NMR Spectroscopy, OxfordUniversity Press; Wüthrich (1986) NMR of Proteins and Nucleic Acids(Baker Lecture Series), Kurt Wiley-Interscience; Neuhaus and Williamson(2000) The Nuclear Overhauser Effect in Structural and ConformationalAnalysis, 2nd Edition, Wiley-VCH; Macomber (1998) A CompleteIntroduction to Modern NMR Spectroscopy, Wiley-Interscience; Downing(2004) Protein NMR Techniques (Methods in Molecular Biology), 2ndedition, Humana Press; Clore and Gronenborn (1994) NMR of Proteins(Topics in Molecular and Structural Biology), CRC Press; Reid (1997)Protein NMR Techniques, Humana Press; Krishna and Berliner (2003)Protein NMR for the Millenium (Biological Magnetic Resonance), KluwerAcademic Publishers; Kiihne and De Groot (2001) Perspectives on SolidState NMR in Biology (Focus on Structural Biology, 1), Kluwer AcademicPublishers; Jones et al. (1993) Spectroscopic Methods and Analyses: NMR,Mass Spectrometry, and Related Techniques (Methods in Molecular Biology,Vol. 17), Humana Press; Goto and Kay (2000) Curr. Opin. Struct. Biol.10:585; Gardner (1998) Annu. Rev. Biophys. Biomol. Struct. 27:357;Wüthrich (2003) Angew. Chem. Int. Ed. 42:3340; Bax (1994) Curr. Opin.Struct. Biol. 4:738; Pervushin et al. (1997) Proc. Natl. Acad. Sci.U.S.A. 94:12366; Fiaux et al. (2002) Nature 418:207; Fernandez and Wider(2003) Curr. Opin. Struct. Biol. 13:570; Ellman et al. (1992) J. Am.Chem. Soc. 114:7959; Wider (2000) BioTechniques 29:1278-1294; Pellecchiaet al. (2002) Nature Rev. Drug Discov. (2002) 1:211-219; Arora and Tamm(2001) Curr. Opin. Struct. Biol. 11:540-547; Flaux et al. (2002) Nature418:207-211; Pellecchia et al. (2001) J. Am. Chem. Soc. 123:4633-4634;and Pervushin et al. (1997) Proc. Natl. Acad. Sci. USA 94:12366-12371.

The structure of a polymerase or of a polymerase bound to a DNA or witha given nucleotide analog incorporated into the active site can, asnoted, be directly determined, e.g., by x-ray crystallography or NMRspectroscopy, or the structure can be modeled based on the structure ofthe polymerase and/or a structure of a polymerase with a naturalnucleotide bound. The active site or other relevant domain of thepolymerase can be identified, for example, by homology with otherpolymerases, examination of polymerase-template or polymerase-nucleotideco-complexes, biochemical analysis of mutant polymerases, and/or thelike. The position of a nucleotide analog (as opposed to an availablenucleotide structure) in the active site can be modeled, for example, byprojecting the location of non-natural features of the analog (e.g.,additional phosphate or phosphonate groups in the phosphorus containingchain linked to the nucleotide, e.g., tetra, penta or hexa phosphategroups, detectable labeling groups, e.g., fluorescent dyes, or the like)based on the previously determined location of another nucleotide ornucleotide analog in the active site.

Such modeling of the nucleotide analog or template (or both) in theactive site can involve simple visual inspection of a model of thepolymerase, for example, using molecular graphics software such as thePyMOL viewer (open source, freely available on the World Wide Web atwww(dot)pymol(dot)org), Insight II, or Discovery Studio 2.1(commercially available from Accelrys at (www (dot) accelrys (dot)com/products/discovery-studio). Alternatively, modeling of the activesite complex of the polymerase or a putative mutant polymerase, forexample, can involve computer-assisted docking, molecular dynamics, freeenergy minimization, and/or like calculations. Such modeling techniqueshave been well described in the literature; see, e.g., Babine andAbdel-Meguid (eds.) (2004) Protein Crystallography in Drug Design,Wiley-VCH, Weinheim; Lyne (2002) “Structure-based virtual screening: Anoverview” Drug Discov. Today 7:1047-1055; Molecular Modeling forBeginners, at (www (dot) usm (dot) maine (dot) edu/˜rhodes/SPVTut/index(dot) html; and Methods for Protein Simulations and Drug Design at (www(dot) dddc (dot) ac (dot) cn/embo04; and references therein. Software tofacilitate such modeling is widely available, for example, the CHARMmsimulation package, available academically from Harvard University orcommercially from Accelrys (at www (dot) accelrys (dot) com), theDiscover simulation package (included in Insight II, supra), and Dynama(available at (www(dot) cs (dot) gsu (dot) edu/˜cscrwh/progs/progs (dot)html). See also an extensive list of modeling software at (www (dot)netsci (dot) org/Resources/Software/Modeling/MMMD/top (dot) html.

Visual inspection and/or computational analysis of a polymerase model,including optional comparison of models of the polymerase in differentstates, can identify relevant features of the polymerase, including, forexample, residues that can be mutated to increase stability orreadlength, as detailed above.

In another example, residues from domains that are in close proximity toone another are mutated to alter inter-domain interactions. As shown inFIG. 6, in Φ29, M429 is in the interface between the fingers domain andthe rest of the polymerase. Without limitation to any particularmechanism, mutating M429 can modulate the movement of the fingers domainand alter the equilibrium between the binary and ternary complexes. AnM429A substitution, for example, can narrow pulse width and speed thepolymerase.

Amino acid sequence data, e.g., for members of a family of polymerases,can be used in conjunction with structural data to identify particularresidues as candidates for mutagenesis. As one example, residues thatdiffer between family members and that are close to the active site canbe mutated. For example, as shown in FIG. 1, wild-type Φ29 has analanine at position 256 while wild-type M2Y has a serine at thecorresponding position (position 253 of M2Y, SEQ ID NO:2). Introducingan S253A substitution into M2Y, where positions are numbered withrespect to SEQ ID NO:2, can increase readlength and decrease pulsewidth. An A256S substitution can be introduced into Φ29, where positionsare numbered with respect to SEQ ID NO:1, e.g., to increase pulse width.As another example, wild-type Φ29 has a glutamic acid at position 175while wild-type M2Y has an arginine at the corresponding position(position 172 of M2Y, SEQ ID NO:2). An E175R substitution can beintroduced into Φ29, where positions are numbered with respect to SEQ IDNO:1, or an R172E substitution can be introduced into M2Y, wherepositions are numbered with respect to SEQ ID NO:2. As yet anotherexample, wild-type Φ29 has a tyrosine at position 224 while wild-typeM2Y has a lysine at the corresponding position (position 221 of M2Y, SEQID NO:2). A Y224K substitution can be introduced into Φ29, wherepositions are numbered with respect to SEQ ID NO:1, or a K221Ysubstitution can be introduced into M2Y, where positions are numberedwith respect to SEQ ID NO:2. As yet another example, wild-type Φ29 has amethionine at position 506 while wild-type M2Y has a valine at thecorresponding position (position 503 of M2Y, SEQ ID NO:2). A V503Msubstitution can be introduced into M2Y, where positions are numberedwith respect to SEQ ID NO:2. As yet another example, wild-type Φ29 hasan aspartic acid at position 570 and a threonine at position 571 whilewild-type M2Y has a serine and a valine at the corresponding positions(positions 567 and 568 of M2Y, SEQ ID NO:2). A D570S and/or a T571Vsubstitution can be introduced into Φ29, where positions are numberedwith respect to SEQ ID NO:1, e.g., to increase processivity(particularly when D570S and T571V are employed in combination with eachother).

Combining Mutations

As noted repeatedly, the various mutations described herein can becombined in recombinant polymerases of the invention. Combination ofmutations can be random, or more desirably, guided by the properties ofthe particular mutations and the characteristics desired for theresulting polymerase. Additional mutations can also be introduced into apolymerase to compensate for deleterious effects of otherwise desirablemutations.

A large number of exemplary mutations and the properties they confer aredescribed herein, and it will be evident that these mutations can befavorably combined in many different combinations. Exemplarycombinations are also provided herein, e.g., in Tables 3-4 and 7-8 andFIGS. 7-9, and an example of strategies by which additional favorablecombinations are readily derived follows. For the sake of simplicity, afew exemplary combinations using only a few exemplary mutations arediscussed, but it will be evident that any of the mutations describedherein can be employed in such strategies to produce polymerases withdesirable properties.

For example, where a recombinant polymerase is desired to incorporatephosphate-labeled phosphate analogs and/or large analogs in aMg⁺⁺-containing single molecule sequencing reaction, one or moresubstitutions that enhance analog binding (e.g., E375Y, K512Y, and/orA484E) and one or more substitutions that alter metal cofactor usage(e.g., L253A, L253H, or L253S) can be incorporated. Polymerase speed canbe enhanced by inclusion of substitutions such as A437G, E508K, V141K,L142K, D510K, and/or V250I. Accuracy can be enhanced by inclusion ofsubstitutions such as Q99Y, E515Q, D235E, Y148I, A134S, K536T, and/orK536E. (A Q99Y, A134S, K536T, or K536E substitution can decrease thefrequency of certain noncognate errors in which a G nucleotide or analogbinds to a template G, producing a spurious pulse and thus an insertionerror. Such errors can be more common when large analogs are employed.)Processivity can be increased by inclusion of substitutions such asK138Q, K138A, K138C, D570S, and/or T571V. Stability and/or yield can beincreased by inclusion of substitutions such as E239G, V250I, Y224K,D570E, Y148I, K131E, and/or Q99I, producing combinations such as D570Eand Y148I; Y224K, E239G, L253H, E375Y, A437G, A484E, D510K, K512Y, andE515Q; Y224K, E239G, V250I, L253H, E375Y, A437G, A484E, D510K, K512Y,and E515Q; and Y148I, Y224K, E239G, L253H, E375Y, A437G, A484E, D510K,K512Y, and E515Q. Stability can also be increased, e.g., by employingM2Y as the parental polymerase and/or including a stability-enhancingexogenous feature (e.g., a C-terminal exogenous feature, e.g., a His10or other polyhistidine tag).

One or more substitutions that increase phototolerance (e.g., K131E,K131Q, and/or K135Q) can be included, providing combinations such asK131E, Y224K, E239G, L253H, E375Y, A437G, A484E, D510K, K512Y, andE515Q. Substitutions that reduce photodamage in some instances alsoundesirably increase interpulse distances, so a substitution thatdecreases interpulse distance can be added, providing combinations suchas K131E and E508R, K135Q and E508R, and K131E, K135Q and E508R.

As described above, as another route to reducing photodamage to thepolymerase, one or more nucleotide analogs designed to reducephotodamage, e.g., by increasing the effective distance the dye moietyis maintained from the surface of the polymerase or by otherwiseshielding the polymerase from collision with the dye, can be employed.(See, e.g., U.S. patent application 61/599,149 and U.S. patentapplication Ser. No. 13/767,619 “Polymerase Enzyme Substrates withProtein Shield,” which describe protein-shielded analogs that includestreptavidin, ubiquitin, or the like, U.S. patent application61/862,502, which describes additional shielded analogs, and U.S. Pat.No. 7,968,702, which describes analogs in which the label is maintainedat least 2 nm from the polymerase.) The polymerase optionally includesone or more substitutions to enhance performance of the polymerase withsuch analogs. For example, use of such large analogs can undesirablynarrow pulse width and increase interpulse distance, so one or moresubstitutions that increase pulse width (e.g., P558A, P558F, A256Sand/or S487A) or that decrease interpulse distance or reduce pausing(e.g., Q99P, V141K, L142K, R306Q, R308L, K311E, T441I, E466K, D476H,E508R, L513K, and/or D523R) can be included in the polymerase, providingcombinations such as L142K, D570S and T571V; L142K, E508R, D570S andT571V; L142K, E508R, P558A, D570S and T571V; L142K, E508R, D523R, P558A,D570S and T571V; L142K and P558A; A256S and S487A; R306Q and R308L;V141K, L142K, A256S, R306Q, R308L, T441I, and E508R; and V141K, L142K,A256S, T441I, and E508R.

Exemplary polymerases for use in single molecule sequencing using largenucleotide analogs, such as the protein shield analogs described in U.S.patent application 61/599,149 and U.S. patent application Ser. No.13/767,619 “Polymerase Enzyme Substrates with Protein Shield,” theshielded analogs described in U.S. patent application 61/862,502, orother analogs with a molecular weight of at least 10,000, can includecombinations of substitutions such as, e.g., V141K, L142K, Y224K, E239G,V250I, L253A, A256S, R306Q, R308L, K311E, E375Y, A437G, T441I, A484E,S487A, E508R, D510K, K512Y, and E515Q; Y224K, E239G, V250I, L253A,A256S, E375Y, A437G, A484E, S487A, D510K, K512Y, and E515Q; V141K,L142K, Y224K, E239G, V250I, L253A, A256S, E375Y, A437G, T441I, A484E,S487A, E508R, D510K, K512Y, E515Q, and D570E; A134S, Y224K, E239G,V250I, L253A, A256S, E375Y, A437G, A484E, S487A, D510K, K512Y, andE515Q; Y224K, E239G, V250I, L253A, E375Y, A437G, A484E, E508R, D510K,K512Y, E515Q, and D570M; A134S, Y224K, E239G, V250I, L253A, E375Y,A437G, A484E, S487A, E508R, D510K, K512Y, E515Q, I524S, F526L, andD570E; L142K, Y224K, E239G, V250I, L253A, E375Y, A437G, D476H, A484E,E508R, D510K, K512Y, L513K, E515Q, and D570E; L142K, Y224K, E239G,V250I, L253A, R306Q, R308L, E375Y, A437G, D476H, A484E, E508R, D510K,K512Y, L513K, E515Q, and D570E; L142K, Y224K, E239G, V250I, L253A,R306Q, R308L, E375Y, A437G, E466K, D476H, A484E, E508R, D510K, K512Y,L513K, E515Q, and D570E; L142K, Y224K, E239G, V250I, L253A, R306Q,R308L, E375Y, A437G, E466K, D476H, A484E, E508R, D510K, K512Y, E515Q,and D570E; L142K, Y224K, E239G, V250I, L253A, E375Y, A437G, E466K,D476H, A484E, E508R, D510K, K512Y, E515Q, and D570E; L142K, Y224K,E239G, V250I, L253A, E375Y, M429A, A437G, A484E, E508R, D510K, K512Y,E515Q, and D570E; A134S, L142K, Y224K, E239G, V250I, L253A, E375Y,A437G, A484E, S487A, E508R, D510K, K512Y, E515Q, I524S, F526L, andD570E; Y224K, E239G, V250I, L253A, E375Y, A437G, A484E, S487A, E508R,D510K, K512Y, E515Q, I524S, F526L, and D570M; C106S, E239G, V250I,L253A, E375Y, A437G, A484E, E508R, D510K, K512Y, and E515Q; L142K,Y224K, D235E, E239G, V250A, L253H, E375Y, A437G, A484E, E508R, D510K,K512Y, E515Q, and D570E; Y224K, E239G, V250I, L253A, E375Y, A437G,A484E, E508R, D510K, K512Y, and E515Q; V141K, L142K, Y224K, E239G,V250I, L253A, E375Y, A437G, A484E, E508R, D510K, K512Y, and E515Q;K135Q, L142K, Y224K, E239G, V250I, L253A, R306Q, R308L, E375Y, A437G,E466K, D476H, A484E, E508R, D510R, K512Y, E515Q, D570S, and T571V;K131E, K135Q, L142K, Y224K, E239G, V250I, L253A, E375Y, A437G, A484E,E508R, D510R, K512Y, E515Q, D523R, P558A, D570S, and T571V; A49E, C106S,K114R, K131E, K135Q, L142K, Y224K, E239G, V250I, L253A, Y369E, E375Y,A437G, D476H, A484E, E508R, D510K, K512Y, E515Q, D523R, D570S, andT571V; K135Q, K138Q, L142K, Y224K, E239G, V250I, L253A, R306Q, R308L,E375Y, A437G, E466K, D476H, A484E, E508R, D510K, K512Y, E515Q, I524T,P558A, D570S, and T571V; K135Q, K138Q, L142K, Y224K, E239G, V250I,L253A, R306Q, R308L, E375Y, A437G, E466K, V475I, D476H, A484E, E508R,D510K, K512Y, E515Q, I524T, P558A, D570S, and T571V; K135Q, K138Q,L142K, Y224K, E239G, V250I, L253A, R306Q, R308L, T368S, E375Y, A437G,E466K, D476H, A484E, E508R, D510K, K512Y, E515Q, I524T, P558A, D570S,and T571V; K135Q, K138Q, L142K, Y224K, E239G, V250I, L253A, R306Q,R308L, T368S, E375Y, A437G, E466K, V475I, D476H, A484E, E508R, D510K,K512Y, E515Q, P558A, D570S, and T571V; L44A, K135Q, K138Q, L142K, Y224K,E239G, V250I, L253A, R306Q, R308L, E375Y, A437G, E466K, D476H, A484E,S487A, E508R, D510K, K512Y, E515Q, P558A, D570S, and T571V; K135Q,L142K, Y224K, E239G, V250I, L253A, R306Q, R308L, T368S, E375Y, A437G,E466K, D476H, A484E, E508R, D510R, K512Y, E515Q, P558A, D570S, andT571V; A49E, C106S, K114R, K135Q, L142K, Y224K, E239G, V250I, L253A,R306Q, R308L, E375Y, A437G, E466K, D476H, A484E, E508R, D510R, K512Y,E515Q, K536Q, K539Q, D570S, and T571V; L44A, K135Q, K138Q, L142K, Y224K,E239G, V250I, L253A, R306Q, R308L, T368S, E375Y, A437G, E466K, D476H,A484E, E508R, D510K, K512Y, E515Q, P558A, D570S, and T571V; L142K,Y224K, E239G, V250I, L253A, R306Q, R308L, E375Y, A437G, E466K, D476H,A484E, E508R, D510K, K512Y, E515Q, P558A, D570T, and T571A; K138C,L142K, Y224K, E239G, V250I, L253A, R306Q, R308L, E375Y, A437G, E466K,D476H, A484E, E508R, D510K, K512Y, E515Q, D570S, and T571V; K138Q,L142K, Y224K, E239G, V250I, L253A, R306Q, R308L, E375Y, A437G, E466K,D476H, A484E, E508R, D510K, K512Y, E515Q, D570S, and T571V; K135Q,L142K, Y224K, E239G, V250I, L253A, R306Q, R308L, E375Y, A437G, E466K,D476H, A484E, E508R, D510R, K512Y, E515Q, K539E, D570S, and T571V;K131E, K135Q, L142K, Y224K, E239G, V250I, L253A, E375Y, A437G, D476H,A484E, E508R, D510K, K512Y, E515Q, D523R, D570S, and T571V; and L142K,Y224K, E239G, V250I, L253A, R306Q, R308L, E375Y, A437G, E466K, D476H,A484E, E508R, D510K, K512Y, E515Q, and D570M.

Detection of base modifications (e.g., 5-methylcytosine, e.g., asdescribed in WO 2012/065043 “Classification of Nucleic Acid Templates”and U.S. patent application 61/617,999, filed Mar. 30, 2012 and entitled“Methods and Compositions for Sequencing Modified Nucleic Acids”) can beenhanced by inclusion of substitutions such as Q99W, producingcombinations such as Q99W, Y224K, E239G, L253H, E375Y, A437G, A484E,D510K, K512Y, and E515Q; Q99W, K131E, Y224K, E239G, L253H, E375Y, A437G,A484E, D510K, K512Y, and E515Q; Q99W, Y148I, Y224K, E239G, L253H, E375Y,A437G, A484E, D510K, K512Y, and E515Q; Q99W, Y224K, E239G, V250I, L253H,E375Y, A437G, A484E, D510K, K512Y, and E515Q; Q99W, K131E, Y148I, Y224K,E239G, V250I, L253H, E375Y, A437G, A484E, D510K, K512Y, and E515Q; andQ99W, K131E, Y148I, Y224K, E239G, V250I, L253A, E375Y, A437G, A484E,D510K, K512Y, and E515Q.

For applications involving nucleic acid amplification at elevatedtemperature, suitable polymerases typically include mutations thatenhance stability (e.g., polymerase stability, binary complex stability,and/or ternary complex stability). Thus, a polymerase suitable forperforming whole genome amplification can include, e.g., substitutionsthat increase binary complex stability, e.g., D570E, Y148I, and/orK131E. The polymerase can also include substitutions that increasestability and/or yield of the free polymerase, e.g., Y224K, E239G,and/or F526L, providing combinations such as K131E, Y148I, Y224K, andD570E. The polymerase can be derived from an M2Y parental polymerase tofurther enhance stability and/or can include a stability-enhancingexogenous feature (e.g., a C-terminal exogenous feature, e.g., a His10or other polyhistidine tag). The polymerase can include a substitutionthat can enhance its ability to read through sites of DNA damage, e.g.,L253A, providing combinations such as K131E, Y148I, Y224K, L253A, andD570E. A polymerase suitable for performing single molecule sequencingat higher temperature (e.g., 30° C. and above) can include suchstability-enhancing features (e.g., substitutions that increasepolymerase, binary complex, and/or ternary complex stability, aC-terminal tag, and/or an M2Y parental polymerase) as well as mutationsthat broaden pulse width to facilitate detection of nucleotideincorporation events, for example, a combination such as K131E, Y224K,E239G, V250I, L253A, A256S, E375Y, A437G, C455A, A484E, D510K, K512Y,E515Q, F526L, and D570E.

It will be evident that different polymerase properties, and thereforedifferent combinations of mutations, are desirable for differentapplications involving recombinant polymerases. For example, for certainsequencing applications, accuracy can be emphasized. Suitable exemplarycombinations of mutations for use in such polymerases include, e.g.,Q99I, K131E, A134S, Y224K, E239G, V250I, L253A, E375Y, A437N, A484E,E508R, D510K, K512Y, E515Q, and D570E; Q99I, K131E, A134S, Y224K, E239G,V250I, L253A, R306Q, E375Y, A437N, A484E, E508R, D510K, K512Y, E515Q,and D570E; K131E, A134S, Y224K, E239G, V250I, L253A, E375Y, A437N,A484E, E508R, D510K, K512Y, E515Q, and D570E; K131E, A134S, Y148I,Y224K, D235E, E239G, L253H, E375Y, A437G, A484E, D510K, K512Y, andE515Q; and K131E, Y148I, E239G, V250I, L253A, E375Y, A437G, A484E,D510K, K512Y, and E515Q (optionally with an alanine at position 256).

In other sequencing applications such as scaffolding for genome assemblyor finishing (see, e.g., Koren et al. (2012) “Hybrid error correctionand de novo assembly of single-molecule sequencing reads” NatBiotechnol. 30(7):693-700), readlength can be optimized. Exemplarycombinations of substitutions suitable for such applications include,e.g., K131E, L142K, Y224K, D235E, E239G, V250A, L253H, E375Y, A437G,A484E, E508R, D510K, K512Y, and E515Q; K131E, Y224K, E239G, V250I,L253A, E375Y, A437G, C455A, A484E, D510K, K512Y, and E515Q; K131E,Y224K, E239G, L253H, E375Y, M429A, A437G, C455A, A484E, D510K, K512Y,and E515Q; C11A, C106S, K131E, Y224K, E239G, L253H, C290F, K337C, E375Y,A437G, C448V, A484E, D510K, K512Y, and E515Q; and K131E, Y148I, Y224K,E239G, V250I, L253A, E375Y, M429A, A437G, A484E, D510K, K512Y, andE515Q.

Many other such recombinant polymerases, including these mutationsand/or those described elsewhere herein, will be readily apparent andare features of the invention.

Mutating Polymerases

Various types of mutagenesis are optionally used in the presentinvention, e.g., to modify polymerases to produce variants, e.g., inaccordance with polymerase models and model predictions as discussedabove, or using random or semi-random mutational approaches. In general,any available mutagenesis procedure can be used for making polymerasemutants. Such mutagenesis procedures optionally include selection ofmutant nucleic acids and polypeptides for one or more activity ofinterest (e.g., enhanced performance with large nucleotide analogs(e.g., protein shield analogs), increased thermostability, increasedreadlength, increased sensitivity to base modifications, increasedphototolerance, reduced reaction rates, decreased exonuclease activity,increased complex stability, decreased branching fraction, altered metalcofactor selectivity, improved processivity, increased yield, increasedaccuracy, and/or improved k_(off), K_(m), V_(max), k_(cat) etc., e.g.,for a given nucleotide analog). Procedures that can be used include, butare not limited to: site-directed point mutagenesis, random pointmutagenesis, in vitro or in vivo homologous recombination (DNA shufflingand combinatorial overlap PCR), mutagenesis using uracil containingtemplates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA, point mismatch repair, mutagenesis using repair-deficienthost strains, restriction-selection and restriction-purification,deletion mutagenesis, mutagenesis by total gene synthesis, degeneratePCR, double-strand break repair, and many others known to persons ofskill. The starting polymerase for mutation can be any of those notedherein, including available polymerase mutants such as those identifiede.g., in WO 2007/076057 “Polymerases for Nucleotide AnalogueIncorporation” by Hanzel et al.; WO 2008/051530 “Polymerase Enzymes andReagents for Enhanced Nucleic Acid Sequencing”; US patent applicationpublication 2010-0075332 “Engineering Polymerases and ReactionConditions for Modified Incorporation Properties” by Pranav Patel etal.; US patent application publication 2010-0093555 “Enzymes Resistantto Photodamage” by Keith Bjornson et al.; US patent applicationpublication 2010-0112645 “Generation of Modified Polymerases forImproved Accuracy in Single Molecule Sequencing” by Sonya Clark et al.;US patent application publication 2011-0189659 “Generation of ModifiedPolymerases for Improved Accuracy in Single Molecule Sequencing” bySonya Clark et al.; US patent application publication 2012-0034602“Recombinant Polymerases For Improved Single Molecule Sequencing”; U.S.patent application Ser. No. 13/756,113 filed Jan. 31, 2013 by SatwikKamtekar et al. and entitled “Recombinant Polymerases with IncreasedPhototolerance”; Hanzel et al. WO 2007/075987 “Active Surface CoupledPolymerases”; and Hanzel et al. 2007/075873 “Protein EngineeringStrategies to Optimize Activity of Surface Attached Proteins.”

Optionally, mutagenesis can be guided by known information from anaturally occurring polymerase molecule, or of a known altered ormutated polymerase (e.g., using an existing mutant polymerase as notedin the preceding references), e.g., sequence, sequence comparisons,physical properties, crystal structure and/or the like as discussedabove. However, in another class of embodiments, modification can beessentially random (e.g., as in classical or “family” DNA shuffling,see, e.g., Crameri et al. (1998) “DNA shuffling of a family of genesfrom diverse species accelerates directed evolution” Nature391:288-291).

Additional information on mutation formats is found in: Sambrook et al.,Molecular Cloning—A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 2000 (“Sambrook”); CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (supplemented through 2012) (“Ausubel”));and PCR Protocols A Guide to Methods and Applications (Innis et al. eds)Academic Press Inc. San Diego, Calif. (1990) (“Innis”). The followingpublications and references cited within provide additional detail onmutation formats: Arnold, Protein engineering for unusual environments,Current Opinion in Biotechnology 4:450-455 (1993); Bass et al., MutantTrp repressors with new DNA-binding specificities, Science 242:240-245(1988); Bordo and Argos (1991) Suggestions for “Safe” ResidueSubstitutions in Site-directed Mutagenesis 217:721-729; Botstein &Shortle, Strategies and applications of in vitro mutagenesis, Science229:1193-1201 (1985); Carter et al., Improved oligonucleotidesite-directed mutagenesis using M13 vectors, Nucl. Acids Res. 13:4431-4443 (1985); Carter, Site-directed mutagenesis, Biochem. J. 237:1-7(1986); Carter, Improved oligonucleotide-directed mutagenesis using M13vectors, Methods in Enzymol. 154: 382-403 (1987); Dale et al.,Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod, Methods Mol. Biol. 57:369-374 (1996); Eghtedarzadeh & Henikoff,Use of oligonucleotides to generate large deletions, Nucl. Acids Res.14: 5115 (1986); Fritz et al., Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro, Nucl. Acids Res. 16: 6987-6999 (1988); Grundström et al.,Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’ genesynthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Hayes (2002) CombiningComputational and Experimental Screening for rapid Optimization ofProtein Properties PNAS 99(25) 15926-15931; Kunkel, The efficiency ofoligonucleotide directed mutagenesis, in Nucleic Acids & MolecularBiology (Eckstein, F. and Lilley, D. M. J. eds., Springer Verlag,Berlin)) (1987); Kunkel, Rapid and efficient site-specific mutagenesiswithout phenotypic selection, Proc. Natl. Acad. Sci. USA 82:488-492(1985); Kunkel et al., Rapid and efficient site-specific mutagenesiswithout phenotypic selection, Methods in Enzymol. 154, 367-382 (1987);Kramer et al., The gapped duplex DNA approach tooligonucleotide-directed mutation construction, Nucl. Acids Res. 12:9441-9456 (1984); Kramer & Fritz Oligonucleotide-directed constructionof mutations via gapped duplex DNA, Methods in Enzymol. 154:350-367(1987); Kramer et al., Point Mismatch Repair, Cell 38:879-887 (1984);Kramer et al., Improved enzymatic in vitro reactions in the gappedduplex DNA approach to oligonucleotide-directed construction ofmutations, Nucl. Acids Res. 16: 7207 (1988); Ling et al., Approaches toDNA mutagenesis: an overview, Anal Biochem. 254(2): 157-178 (1997);Lorimer and Pastan Nucleic Acids Res. 23, 3067-8 (1995); Mandecki,Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis, Proc. Natl.Acad. Sci. USA, 83:7177-7181 (1986); Nakamaye & Eckstein, Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 14: 9679-9698 (1986); Nambiar et al., Total synthesis and cloningof a gene coding for the ribonuclease S protein, Science 223: 1299-1301(1984); Sakamar and Khorana, Total synthesis and expression of a genefor the a-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin), Nucl. Acids Res. 14: 6361-6372 (1988); Sayers etal., Y-T Exonucleases in phosphorothioate-based oligonucleotide-directedmutagenesis, Nucl. Acids Res. 16:791-802 (1988); Sayers et al., Strandspecific cleavage of phosphorothioate-containing DNA by reaction withrestriction endonucleases in the presence of ethidium bromide, (1988)Nucl. Acids Res. 16: 803-814; Sieber, et al., Nature Biotechnology,19:456-460 (2001); Smith, In vitro mutagenesis, Ann. Rev. Genet.19:423-462 (1985); Methods in Enzymol. 100: 468-500 (1983); Methods inEnzymol. 154: 329-350 (1987); Stemmer, Nature 370, 389-91 (1994); Tayloret al., The use of phosphorothioate-modified DNA in restriction enzymereactions to prepare nicked DNA, Nucl. Acids Res. 13: 8749-8764 (1985);Taylor et al., The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA, Nucl.Acids Res. 13: 8765-8787 (1985); Wells et al., Importance ofhydrogen-bond formation in stabilizing the transition state ofsubtilisin, Phil. Trans. R. Soc. Lond. A 317: 415-423 (1986); Wells etal., Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites, Gene 34:315-323 (1985); Zoller &Smith, Oligonucleotide-directed mutagenesis using M13-derived vectors:an efficient and general procedure for the production of point mutationsin any DNA fragment, Nucleic Acids Res. 10:6487-6500 (1982); Zoller &Smith, Oligonucleotide-directed mutagenesis of DNA fragments cloned intoM13 vectors, Methods in Enzymol. 100:468-500 (1983); Zoller & Smith,Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template, Methods inEnzymol. 154:329-350 (1987); Clackson et al. (1991) “Making antibodyfragments using phage display libraries” Nature 352:624-628; Gibbs etal. (2001) “Degenerate oligonucleotide gene shuffling (DOGS): a methodfor enhancing the frequency of recombination with family shuffling” Gene271:13-20; and Hiraga and Arnold (2003) “General method forsequence-independent site-directed chimeragenesis: J. Mol. Biol.330:287-296. Additional details on many of the above methods can befound in Methods in Enzymology Volume 154, which also describes usefulcontrols for trouble-shooting problems with various mutagenesis methods.

Determining Kinetic Parameters

The polymerases of the invention can be screened or otherwise tested todetermine whether the polymerase displays a modified activity for orwith a nucleotide analog or template as compared to a parental DNApolymerase (e.g., a corresponding wild-type or available mutantpolymerase from which the recombinant polymerase of the invention wasderived). For example, branching fraction, a reaction rate constant,k_(off), k_(cat), K_(m), V_(max), k_(cat)/K_(m), V_(max)/K_(m), k_(pol),and/or K_(d) of the recombinant DNA polymerase for the nucleotide (oranalog) or template nucleic acid can be determined. The specificityconstant k_(cat)/K_(m) is also a useful measure, e.g., for assessingbranch rate. k_(cat)/K_(m) is a measure of substrate binding that leadsto product formation (and, thus, includes terms defining binding K_(d)and inversely predicts branching fraction formation).

As is well-known in the art, for enzymes obeying simple Michaelis-Mentenkinetics, kinetic parameters are readily derived from rates of catalysismeasured at different substrate concentrations. The Michaelis-Mentenequation, V=V_(max)[S]([S]+K_(m))⁻¹, relates the concentration of freesubstrate ([S], approximated by the total substrate concentration), themaximal rate (V_(max), attained when the enzyme is saturated withsubstrate), and the Michaelis constant (K_(m), equal to the substrateconcentration at which the reaction rate is half of its maximal value),to the reaction rate (V).

For many enzymes, K_(m) is equal to the dissociation constant of theenzyme-substrate complex and is thus a measure of the strength of theenzyme-substrate complex. For such an enzyme, in a comparison of K_(m)s,a lower K_(m) represents a complex with stronger binding, while a higherK_(m) represents a complex with weaker binding. The ratio k_(cat)/K_(m),sometimes called the specificity constant, can be thought of as thesecond order rate constant times the probability of that substrate beingconverted to product once bound. The larger the specificity constant,the more efficient the enzyme is in binding the substrate and convertingit to product. The specificity constant is inversely proportional to thebranching rate, as branching rate is the rate at which the enzyme bindssubstrate (e.g., nucleotide) but does not convert it to product (e.g., aDNA polymer).

k_(cat) (also called the turnover number of the enzyme) can bedetermined if the total enzyme concentration ([E_(T)], i.e., theconcentration of active sites) is known, since V_(max)=k_(cat)[E_(T)].For situations in which the total enzyme concentration is difficult tomeasure, the ratio V_(max)/K_(m) is often used instead as a measure ofefficiency. K_(m) and V_(max) can be determined, for example, from aLineweaver-Burk plot of 1/V against 1/[S], where the y interceptrepresents 1/V_(max), the x intercept −1/K_(m), and the slopeK_(m)/V_(max), or from an Eadie-Hofstee plot of V against V/[S], wherethe y intercept represents V_(max), the x intercept V_(max)/K_(m), andthe slope −K_(m). Software packages such as KinetAsyst™ or Enzfit(Biosoft, Cambridge, UK) can facilitate the determination of kineticparameters from catalytic rate data.

For enzymes such as polymerases that have multiple substrates, varyingthe concentration of only one substrate while holding the others insuitable excess (e.g., effectively constant) concentration typicallyyields normal Michaelis-Menten kinetics.

Details regarding k_(off) determination are described, e.g., in USpatent application publication 2012-0034602. In general, thedissociation rate can be measured in any manner that detects thepolymerase/DNA complex over time. This includes stopped-flowspectroscopy, or even simply taking aliquots over time and testing forpolymerase activity on the template of interest. Free polymerase iscaptured with a polymerase trap after dissociation, e.g., by incubationin the presence of heparin or an excess of competitor DNA (e.g.,non-specific salmon sperm DNA, or the like).

In one embodiment, using pre-steady-state kinetics, the nucleotideconcentration dependence of the rate constant k_(obs) (the observedfirst-order rate constant for dNTP incorporation) provides an estimateof the K_(m) for a ground state binding and the maximum rate ofpolymerization (k_(pol)). The k_(obs) is measured using a burst assay.The results of the assay are fitted with the Burst equation;Product=A[1−exp(−k_(obs)*t)]+k_(ss)*t where A represents amplitude anestimate of the concentration of the enzyme active sites, k_(ss) is theobserved steady-state rate constant and t is the reaction incubationtime. The K_(m) for dNTP binding to the polymerase-DNA complex and thek_(pol) are calculated by fitting the dNTP concentration dependentchange in the k_(obs) using the equationk_(obs)=(k_(pol)*[S])*(K_(m)+[S])⁻¹ where [S] is the substrateconcentration. Results are optionally obtained from a rapid-quenchexperiment (also called a quench-flow measurement), for example, basedon the methods described in Johnson (1986) “Rapid kinetic analysis ofmechanochemical adenosinetriphosphatases” Methods Enzymol. 134:677-705,Patel et al. (1991) “Pre-steady-state kinetic analysis of processive DNAreplication including complete characterization of anexonuclease-deficient mutant” Biochemistry 30(2):511-25, and Tsai andJohnson (2006) “A new paradigm for DNA polymerase specificity”Biochemistry 45(32):9675-87.

Parameters such as rate of binding of a nucleotide analog or template bythe recombinant polymerase, rate of product release by the recombinantpolymerase, or branching rate of the recombinant polymerase can also bedetermined, and optionally compared to that of a parental polymerase(e.g., a corresponding wild-type polymerase).

For a more thorough discussion of enzyme kinetics, see, e.g., Berg,Tymoczko, and Stryer (2002) Biochemistry, Fifth Edition, W. H. Freeman;Creighton (1984) Proteins: Structures and Molecular Principles, W. H.Freeman; and Fersht (1985) Enzyme Structure and Mechanism, SecondEdition, W. H. Freeman.

In one aspect, the improved activity of the enzymes of the invention iscompared with a given parental polymerase. For example, in the case ofenzymes derived from a Φ29 parental enzyme, where the improvement beingsought is an increase in stability of the closed complex, an improvedenzyme of the invention would have a lower k_(off) than the parentalenzyme, e.g., wild type Φ29. Such comparisons are made under equivalentreaction conditions, e.g., equal concentrations of the parental andmodified polymerase, equal substrate concentrations, equivalent solutionconditions (pH, salt concentration, presence of divalent cations, etc.),temperature, and the like. In one aspect, the improved activity of theenzymes of the invention is measured with reference to a model analog oranalog set and compared with a given parental enzyme. Optionally, theimproved activity of the enzymes of the invention is measured underspecified reaction conditions. While the foregoing may be used as acharacterization tool, it in no way is intended as a specificallylimiting reaction of the invention.

Optionally, the polymerase exhibits a K_(m) for a phosphate-labelednucleotide analog that is less than a K_(m) observed for a wild-typepolymerase for the analog to facilitate applications in which thepolymerase incorporates the analog, e.g., during SMS. For example, themodified recombinant polymerase can exhibit a K_(m) for thephosphate-labeled nucleotide analog that is less than less than 75%,50%, 25% or less than that of wild-type or parental polymerase such as awild type Φ29. In one specific class of examples, the polymerases of theinvention have a K_(m) of about 10 μM or less for a non-naturalnucleotide analog such as a phosphate labeled analog.

Screening Polymerases

Screening or other protocols can be used to determine whether apolymerase displays a modified activity, e.g., for a nucleotide analog,as compared to a parental DNA polymerase. For example, branchingfraction, rate constant, k_(off), k_(cat), K_(m), V_(max), ork_(cat)/K_(m) of the recombinant DNA polymerase for the template ornucleotide or analog can be determined as discussed above. As anotherexample, activity can be assayed indirectly. Assays for properties suchas protein yield, thermostability, and the like are described, e.g.,herein and in US patent application publication 2012-0034602.Performance of a recombinant polymerase in a sequencing reaction, e.g.,a single molecule sequencing reaction, can be examined to assayproperties such as speed, pulse width, interpulse distance, accuracy,readlength, ability to incorporate large nucleotide analogs, sensitivityto base modifications, etc. as described herein. Phototolerance can beassessed by monitoring polymerase performance (e.g., in a singlemolecule sequencing reaction) during or after exposure of the polymeraseto light, e.g., excitation light of a specified wavelength at a givenintensity for a given time, e.g., as compared to a wild-type or otherparental polymerase. Resistance to cosolvents can be assessed bymonitoring polymerase performance in the presence of varying amounts ofthe solvent (e.g., in a primarily aqueous solution containing variousamounts of an organic solvent, e.g., DMSO, e.g., 1-10%, 2-10%, 2-5%, or5-8% by volume of the solvent).

In one desirable aspect, a library of recombinant DNA polymerases can bemade and screened for these properties. For example, a plurality ofmembers of the library can be made to include one or more mutation thatincreases stability, increases readlength, improves detection ofmodified bases, enhances incorporation of large analogs, increasesphototolerance, alters (e.g., decreases) reaction rate constants,improves closed complex stability, decreases branching fraction, alterscofactor selectivity, or increases cosolvent resistance, yield,accuracy, or speed, and/or randomly generated mutations (e.g., wheredifferent members include different mutations or different combinationsof mutations), and the library can then be screened for the propertiesof interest (e.g., increased stability, readlength, utility of largeanalogs, or phototolerance, decreased rate constant, decreased branchingfraction, increased closed complex stability, etc.). In general, thelibrary can be screened to identify at least one member comprising amodified activity of interest.

Libraries of polymerases can be either physical or logical in nature.Moreover, any of a wide variety of library formats can be used. Forexample, polymerases can be fixed to solid surfaces in arrays ofproteins. Similarly, liquid phase arrays of polymerases (e.g., inmicrowell plates) can be constructed for convenient high-throughputfluid manipulations of solutions comprising polymerases. Liquid,emulsion, or gel-phase libraries of cells that express recombinantpolymerases can also be constructed, e.g., in microwell plates, or onagar plates. Phage display libraries of polymerases or polymerasedomains (e.g., including the active site region or interdomain stabilityregions) can be produced. Likewise, yeast display libraries can be used.Instructions in making and using libraries can be found, e.g., inSambrook, Ausubel and Berger, referenced herein.

For the generation of libraries involving fluid transfer to or frommicrotiter plates, a fluid handling station is optionally used. Several“off the shelf” fluid handling stations for performing such transfersare commercially available, including e.g., the Zymate systems fromCaliper Life Sciences (Hopkinton, Mass.) and other stations whichutilize automatic pipettors, e.g., in conjunction with the robotics forplate movement (e.g., the ORCA® robot, which is used in a variety oflaboratory systems available, e.g., from Beckman Coulter, Inc.(Fullerton, Calif.).

In an alternate embodiment, fluid handling is performed in microchips,e.g., involving transfer of materials from microwell plates or otherwells through microchannels on the chips to destination sites(microchannel regions, wells, chambers or the like). Commerciallyavailable microfluidic systems include those fromHewlett-Packard/Agilent Technologies (e.g., the HP2100 bioanalyzer) andthe Caliper High Throughput Screening System. The Caliper HighThroughput Screening System provides one example interface betweenstandard microwell library formats and Labchip technologies. RainDanceTechnologies' nanodroplet platform provides another method for handlinglarge numbers of spatially separated reactions. Furthermore, the patentand technical literature includes many examples of microfluidic systemswhich can interface directly with microwell plates for fluid handling.

Tags and Other Optional Polymerase Features

The recombinant DNA polymerase optionally includes additional featuresexogenous or heterologous to the polymerase. For example, therecombinant polymerase optionally includes one or more tags, e.g.,purification, substrate binding, or other tags, such as a polyhistidinetag, a His10 tag, a His6 tag, an alanine tag, an Ala10 tag, an Ala16tag, a biotin tag, a biotin ligase recognition sequence or other biotinattachment site (e.g., a BiTag or a Btag or variant thereof, e.g.,BtagV1-11), a GST tag, an S Tag, a SNAP-tag, an HA tag, a DSB (Sso7D)tag, a lysine tag, a NanoTag, a Cmyc tag, a tag or linker comprising theamino acids glycine and serine, a tag or linker comprising the aminoacids glycine, serine, alanine and histidine, a tag or linker comprisingthe amino acids glycine, arginine, lysine, glutamine and proline, aplurality of polyhistidine tags, a plurality of His10 tags, a pluralityof His6 tags, a plurality of alanine tags, a plurality of Ala10 tags, aplurality of Ala16 tags, a plurality of biotin tags, a plurality of GSTtags, a plurality of BiTags, a plurality of S Tags, a plurality ofSNAP-tags, a plurality of HA tags, a plurality of DSB (Sso7D) tags, aplurality of lysine tags, a plurality of NanoTags, a plurality of Cmyctags, a plurality of tags or linkers comprising the amino acids glycineand serine, a plurality of tags or linkers comprising the amino acidsglycine, serine, alanine and histidine, a plurality of tags or linkerscomprising the amino acids glycine, arginine, lysine, glutamine andproline, biotin, avidin, an antibody or antibody domain, antibodyfragment, antigen, receptor, receptor domain, receptor fragment, maltosebinding protein, ligand, one or more protease site (e.g., Factor Xa,enterokinase, or thrombin site), a dye, an acceptor, a quencher, a DNAbinding domain (e.g., a helix-hairpin-helix domain from topoisomeraseV), a domain that binds modified bases (e.g., an MeCpG binding protein 2domain, an 06-alkylguanine DNA alkyl transferase domain, a thyminedioxygenase JBP1 catalytic domain, or an SRA domain, e.g., from UHRF1),a sliding clamp domain or the like to increase affinity for DNA (e.g.,an HSV UL42 domain), or combination thereof. See, e.g., US patentapplication publication 2012-0034602 for sequences of a number ofsuitable tags and linkers, including BtagV1-11; see also Table 11hereinbelow. The one or more exogenous or heterologous features can finduse not only for purification purposes, immobilization of the polymeraseto a substrate, and the like, but can also be useful for altering one ormore properties of the polymerase (e.g., addition of an exogenousfeature at the C-terminus (e.g., a His10 or other polyhistidine tag) candecrease exonuclease activity and/or increase binary and/or ternarycomplex stability).

The one or more exogenous or heterologous features can be includedinternal to the polymerase, at the N-terminal region of the polymerase,at the C-terminal region of the polymerase, or at a combination thereof(e.g., at both the N-terminal and C-terminal regions of the polymerase).Where the polymerase includes an exogenous or heterologous feature atboth the N-terminal and C-terminal regions, the exogenous orheterologous features can be the same (e.g., a polyhistidine tag, e.g.,a His10 tag, at both the N- and C-terminal regions) or different (e.g.,a biotin ligase recognition sequence at the N-terminal region and apolyhistidine tag, e.g., His10 tag, at the C-terminal region).Optionally, a terminal region (e.g., the N- or C-terminal region) of apolymerase of the invention can comprise two or more exogenous orheterologous features which can be the same or different (e.g., a biotinligase recognition sequence and a polyhistidine tag at the N-terminalregion, a biotin ligase recognition sequence, a polyhistidine tag, and aFactor Xa recognition site at the N-terminal region, and the like). As afew examples, the polymerase can include a polyhistidine tag at theC-terminal region, a biotin ligase recognition sequence at theN-terminal region and a polyhistidine tag at the C-terminal region, abiotin ligase recognition sequence and a polyhistidine tag at theN-terminal region, a biotin ligase recognition sequence and apolyhistidine tag at the N-terminal region and a polyhistidine tag atthe C-terminal region, two biotin ligase recognition sequences at theC-terminal region (e.g., two tandem sequences, e.g., tandem Btags), or apolyhistidine tag and a biotin ligase recognition sequence at theC-terminal region.

For convenience, an exogenous or heterologous feature will often beexpressed as a fusion domain of the overall polymerase protein, e.g., asa conventional in-frame fusion of a polypeptide sequence with the activepolymerase enzyme (e.g., a polyhistidine tag fused in frame to an activepolymerase enzyme sequence). However, features such as tags can be addedchemically to the polymerase, e.g., by using an available amino acidresidue of the enzyme or by incorporating an amino acid into the proteinthat provides a suitable attachment site for the coupling domain.Suitable residues of the enzyme can include, e.g., histidine, cysteine,or serine residues (providing for N, S, or O linked coupling reactions).Optionally, one or more cysteines present in the parental polymerase(e.g., up to all of the cysteines present on the polymerase's surface)can be replaced with a different amino acid; either a single reactivesurface cysteine can be left unsubstituted or a single reactive surfacecysteine can be introduced in place of another residue, for convenientaddition of a feature, e.g., for surface immobilization through thiollabeling (e.g., addition of maleimide biotin, or maleimide and an alkynefor click labeling). Unnatural amino acids that comprise unique reactivesites can also be added to the enzyme, e.g., by expressing the enzyme ina system that comprises an orthogonal tRNA and an orthogonal synthetasethat loads the unnatural amino acid in response to a selector codon.

The exogenous or heterologous features can find use, e.g., in thecontext of binding a polymerase in an active form to a surface, e.g., toorient and/or protect the polymerase active site when the polymerase isbound to a surface. In general, surface binding elements andpurification tags that can be added to the polymerase (e.g.,recombinantly or chemically) include, e.g., biotin attachment sites(e.g., biotin ligase recognition sequences such as Btags or BiTag),polyhistidine tags, His6 tags, His10 tags, biotin, avidin, GSTsequences, modified GST sequences, e.g., that are less likely to formdimers, S tags, SNAP-tags, antibodies or antibody domains, antibodyfragments, antigens, receptors, receptor domains, receptor fragments,ligands, and combinations thereof.

One aspect of the invention includes DNA polymerases that can be coupledto a surface without substantial loss of activity (e.g., in an activeform). DNA polymerases can be coupled to the surface through a singlesurface coupling domain or through multiple surface coupling domainswhich act in concert to increase binding affinity of the polymerase forthe surface and to orient the polymerase relative to the surface. Forexample, the active site can be oriented distal to the surface, therebymaking it accessible to a polymerase substrate (template, nucleotides,etc.). This orientation also tends to reduce surface denaturationeffects in the region of the active site. In a related aspect, activityof the enzyme can be protected by making the coupling domains large,thereby serving to further insulate the active site from surface bindingeffects. Further details regarding the immobilization of a polymerase toa surface (e.g., the surface of a zero mode waveguide) in an active formare found in WO 2007/075987 “Active Surface Coupled Polymerases” byHanzel et al. and WO 2007/075873 “Protein Engineering Strategies toOptimize Activity of Surface Attached Proteins” by Hanzel et al. Furtherdetails on attaching tags is available in the art. See, e.g., U.S. Pat.Nos. 5,723,584 and 5,874,239 for additional information on attachingbiotinylation peptides to recombinant proteins.

The polymerase immobilized on a surface in an active form can be coupledto the surface through one or a plurality of artificial or recombinantsurface coupling domains as discussed above, and typically displays ak_(cat)/K_(m) (or V_(max)/K_(m)) that is at least about 1%, at leastabout 10%, at least about 25%, at least about 50%, or at least about 75%as high as a corresponding active polymerase in solution.

Exonuclease-Deficient Recombinant Polymerases

Many native DNA polymerases have a proof-reading exonuclease functionwhich can yield substantial data analysis problems in processes thatutilize real time observation of incorporation events as a method ofidentifying sequence information, e.g., single molecule sequencingapplications. Even where exonuclease activity does not introduce suchproblems in single molecule sequencing, reduction of exonucleaseactivity can be desirable since it can increase accuracy (in some casesat the expense of readlength).

Accordingly, recombinant polymerases of the invention optionally includeone or more mutations (e.g., substitutions, insertions, and/ordeletions) relative to the parental polymerase that reduce or eliminateendogenous exonuclease activity. For example, relative to the wild-typeΦ29 DNA polymerase of SEQ ID NO:1, one or more of positions N62, D12,E14, T15, H61, D66, D169, K143, Y148, and H149 is optionally mutated toreduce exonuclease activity. Exemplary mutations that can reduceexonuclease activity include, e.g., N62D, N62H, D12A, T15I, E14I, E14A,D66A, K143D, D145A and D169A substitutions, as well as addition of anexogenous feature at the C-terminus (e.g., a polyhistidine tag).Additional exemplary substitutions in the exonuclease domain includeN62S, D12N, D12R, D12M, E14Q, H61K, H61D, H61A, D66R, D66N, D66Q, D66K,D66M, D169N, K143R, Y148K, Y148A, Y148C, Y148D, Y148E, Y148F, Y148G,Y148H, Y148I, Y148L, Y148M, Y148N, Y148P, Y148Q, Y148R, Y148S, Y148T,Y148V, Y148W, and H149M. The polymerases of the invention optionallycomprise one or more of these mutations. For example, in one aspect, thepolymerase is a Φ29-type polymerase that includes one or more mutationsin the N-terminal exonuclease domain (residues 5-189 as numbered withrespect to wild-type Φ29).

Making and Isolating Recombinant Polymerases

Generally, nucleic acids encoding a polymerase of the invention can bemade by cloning, recombination, in vitro synthesis, in vitroamplification and/or other available methods. A variety of recombinantmethods can be used for expressing an expression vector that encodes apolymerase of the invention. Methods for making recombinant nucleicacids, expression and isolation of expressed products are well known anddescribed in the art. A number of exemplary mutations and combinationsof mutations, as well as strategies for design of desirable mutations,are described herein. Methods for making and selecting mutations in theactive site of polymerases, including for modifying steric features inor near the active site to permit improved access by nucleotide analogsare found hereinabove and, e.g., in WO 2007/076057 “Polymerases forNucleotide Analogue Incorporation” by Hanzel et al. and WO 2008/051530“Polymerase Enzymes and Reagents for Enhanced Nucleic Acid Sequencing”by Rank et al.

Additional useful references for mutation, recombinant and in vitronucleic acid manipulation methods (including cloning, expression, PCR,and the like) include Berger and Kimmel, Guide to Molecular CloningTechniques, Methods in Enzymology volume 152 Academic Press, Inc., SanDiego, Calif. (Berger); Kaufman et al. (2003) Handbook of Molecular andCellular Methods in Biology and Medicine Second Edition Ceske (ed) CRCPress (Kaufman); and The Nucleic Acid Protocols Handbook Ralph Rapley(ed) (2000) Cold Spring Harbor, Humana Press Inc (Rapley); Chen et al.(ed) PCR Cloning Protocols, Second Edition (Methods in MolecularBiology, volume 192) Humana Press; and in Viljoen et al. (2005)Molecular Diagnostic PCR Handbook Springer, ISBN 1402034032.

In addition, a plethora of kits are commercially available for thepurification of plasmids or other relevant nucleic acids from cells,(see, e.g., EasyPrep™, FlexiPrep™ both from Pharmacia Biotech;StrataClean™, from Stratagene; and, QIAprep™ from Qiagen). Any isolatedand/or purified nucleic acid can be further manipulated to produce othernucleic acids, used to transfect cells, incorporated into relatedvectors to infect organisms for expression, and/or the like. Typicalcloning vectors contain transcription and translation terminators,transcription and translation initiation sequences, and promoters usefulfor regulation of the expression of the particular target nucleic acid.The vectors optionally comprise generic expression cassettes containingat least one independent terminator sequence, sequences permittingreplication of the cassette in eukaryotes, or prokaryotes, or both,(e.g., shuttle vectors) and selection markers for both prokaryotic andeukaryotic systems. Vectors are suitable for replication and integrationin prokaryotes, eukaryotes, or both.

Other useful references, e.g. for cell isolation and culture (e.g., forsubsequent nucleic acid isolation) include Freshney (1994) Culture ofAnimal Cells, a Manual of Basic Technique, third edition, Wiley-Liss,New York and the references cited therein; Payne et al. (1992) PlantCell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. NewYork, N.Y.; Gamborg and Phillips (eds) (1995) Plant Cell, Tissue andOrgan Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag(Berlin Heidelberg New York) and Atlas and Parks (eds) The Handbook ofMicrobiological Media (1993) CRC Press, Boca Raton, Fla.

Nucleic acids encoding the recombinant polymerases of the invention arealso a feature of the invention. A particular amino acid can be encodedby multiple codons, and certain translation systems (e.g., prokaryoticor eukaryotic cells) often exhibit codon bias, e.g., different organismsoften prefer one of the several synonymous codons that encode the sameamino acid. As such, nucleic acids of the invention are optionally“codon optimized,” meaning that the nucleic acids are synthesized toinclude codons that are preferred by the particular translation systembeing employed to express the polymerase. For example, when it isdesirable to express the polymerase in a bacterial cell (or even aparticular strain of bacteria), the nucleic acid can be synthesized toinclude codons most frequently found in the genome of that bacterialcell, for efficient expression of the polymerase. A similar strategy canbe employed when it is desirable to express the polymerase in aeukaryotic cell, e.g., the nucleic acid can include codons preferred bythat eukaryotic cell.

A variety of protein isolation and detection methods are known and canbe used to isolate polymerases, e.g., from recombinant cultures of cellsexpressing the recombinant polymerases of the invention. A variety ofprotein isolation and detection methods are well known in the art,including, e.g., those set forth in R. Scopes, Protein Purification,Springer-Verlag, N.Y. (1982); Deutscher, Methods in Enzymology Vol. 182:Guide to Protein Purification, Academic Press, Inc. N.Y. (1990); Sandana(1997) Bioseparation of Proteins, Academic Press, Inc.; Bollag et al.(1996) Protein Methods, 2^(nd) Edition Wiley-Liss, NY; Walker (1996) TheProtein Protocols Handbook Humana Press, NJ, Harris and Angal (1990)Protein Purification Applications: A Practical Approach IRL Press atOxford, Oxford, England; Harris and Angal Protein Purification Methods:A Practical Approach IRL Press at Oxford, Oxford, England; Scopes (1993)Protein Purification: Principles and Practice 3^(rd) Edition SpringerVerlag, NY; Janson and Ryden (1998) Protein Purification: Principles,High Resolution Methods and Applications, Second Edition Wiley-VCH, NY;and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ; and thereferences cited therein. Additional details regarding proteinpurification and detection methods can be found in Satinder Ahuja ed.,Handbook of Bioseparations, Academic Press (2000).

Kits

The present invention also features kits that incorporate thepolymerases of the invention, optionally with additional useful reagentssuch as one or more nucleotides and/or nucleotide analogs, e.g., forsequencing, nucleic acid amplification, or the like. Such kits caninclude the polymerase of the invention packaged in a fashion to enableuse of the polymerase (e.g., the polymerase immobilized in a ZMW array),optionally with a set of different nucleotide analogs of the invention,e.g., those that are analogous to A, T, G, and C, e.g., where one ormore of the analogs comprise a detectable moiety, to permitidentification in the presence of the analogs (e.g., protein shield orother large analogs, e.g., analogs with a molecular weight of at least10,000). Depending upon the desired application, the kits of theinvention optionally include additional reagents, such as naturalnucleotides, a control template, and other reagents, such as buffersolutions and/or salt solutions, including, e.g., divalent metal ionssuch as Ca⁺⁺, Mg⁺⁺, Mn⁺⁺ and/or Fe⁺⁺, and standard solutions, e.g., dyestandards for detector calibration. Such kits also typically includeinstructions for use of the compounds and other reagents in accordancewith the desired application methods, e.g., nucleic acid sequencing,amplification and the like.

Nucleic Acid and Polypeptide Sequences and Variants

As described herein, the invention also features polynucleotidesequences encoding, e.g., a polymerase as described herein. Examples ofpolymerase sequences that include features found herein, e.g., as inTables 3-10, are provided. However, one of skill in the art willimmediately appreciate that the invention is not limited to thespecifically exemplified sequences. For example, one of skill willappreciate that the invention also provides, e.g., many relatedsequences with the functions described herein, e.g., polynucleotides andpolypeptides encoding conservative variants of a polymerase of Tables3-10 or FIGS. 7-9 or any other specifically listed polymerase herein.Combinations of any of the mutations noted herein or combinations of anyof the mutations herein in combination with those noted in otheravailable references relating to improved polymerases, such as Hanzel etal. WO 2007/076057 “Polymerases for Nucleotide Analogue Incorporation”;Rank et al. WO 2008/051530 “Polymerase Enzymes and Reagents for EnhancedNucleic Acid Sequencing”; Hanzel et al. WO 2007/075987 “Active SurfaceCoupled Polymerases”; Hanzel et al. WO 2007/075873 “Protein EngineeringStrategies to Optimize Activity of Surface Attached Proteins”; US patentapplication publication 2010-0075332 “Engineering Polymerases andReaction Conditions for Modified Incorporation Properties” by PranavPatel et al.; US patent application publication 2010-0093555 “EnzymesResistant to Photodamage” by Keith Bjornson et al.; US patentapplication publication 2010-0112645 “Generation of Modified Polymerasesfor Improved Accuracy in Single Molecule Sequencing” by Sonya Clark etal.; US patent application publication 2011-0189659 “Generation ofModified Polymerases for Improved Accuracy in Single MoleculeSequencing” by Sonya Clark et al.; US patent application publication2012-0034602 “Recombinant Polymerases For Improved Single MoleculeSequencing”; and U.S. patent application Ser. No. 13/756,113 filed Jan.31, 2013 by Satwik Kamtekar et al. and entitled “Recombinant Polymeraseswith Increased Phototolerance” are also features of the invention.

Accordingly, the invention provides a variety of polypeptides(polymerases) and polynucleotides (nucleic acids that encodepolymerases). Exemplary polynucleotides of the invention include, e.g.,any polynucleotide that encodes a polymerase of Tables 3-10 or FIGS. 7-9or otherwise described herein. Because of the degeneracy of the geneticcode, many polynucleotides equivalently encode a given polymerasesequence. Similarly, an artificial or recombinant nucleic acid thathybridizes to a polynucleotide indicated above under highly stringentconditions over substantially the entire length of the nucleic acid (andis other than a naturally occurring polynucleotide) is a polynucleotideof the invention. In one embodiment, a composition includes apolypeptide of the invention and an excipient (e.g., buffer, water,pharmaceutically acceptable excipient, etc.). The invention alsoprovides an antibody or antisera specifically immunoreactive with apolypeptide of the invention (e.g., that specifically recognizes afeature of the polymerase that confers decreased branching or increasedcomplex stability).

In certain embodiments, a vector (e.g., a plasmid, a cosmid, a phage, avirus, etc.) comprises a polynucleotide of the invention. In oneembodiment, the vector is an expression vector. In another embodiment,the expression vector includes a promoter operably linked to one or moreof the polynucleotides of the invention. In another embodiment, a cellcomprises a vector that includes a polynucleotide of the invention.

One of skill will also appreciate that many variants of the disclosedsequences are included in the invention. For example, conservativevariations of the disclosed sequences that yield a functionally similarsequence are included in the invention. Variants of the nucleic acidpolynucleotide sequences, wherein the variants hybridize to at least onedisclosed sequence, are considered to be included in the invention.Unique subsequences of the sequences disclosed herein, as determined by,e.g., standard sequence comparison techniques, are also included in theinvention.

Conservative Variations

Owing to the degeneracy of the genetic code, “silent substitutions”(i.e., substitutions in a nucleic acid sequence which do not result inan alteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence that encodes an amino acid sequence. Similarly,“conservative amino acid substitutions,” where one or a limited numberof amino acids in an amino acid sequence (other than residues noted,e.g., in Tables 3-10 and FIGS. 7-9 or elsewhere herein, as beingrelevant to a feature or property of interest) are substituted withdifferent amino acids with highly similar properties, are also readilyidentified as being highly similar to a disclosed construct. Suchconservative variations of each disclosed sequence are a feature of thepresent invention.

“Conservative variations” of a particular nucleic acid sequence refersto those nucleic acids which encode identical or essentially identicalamino acid sequences, or, where the nucleic acid does not encode anamino acid sequence, to essentially identical sequences. One of skillwill recognize that individual substitutions, deletions or additionswhich alter, add or delete a single amino acid or a small percentage ofamino acids (typically less than 5%, more typically less than 4%, 2% or1%) in an encoded sequence are “conservatively modified variations”where the alterations result in the deletion of an amino acid, additionof an amino acid, or substitution of an amino acid with a chemicallysimilar amino acid, while retaining the relevant mutational feature (forexample, the conservative substitution can be of a residue distal to theactive site region, or distal to an interdomain stability region). Thus,“conservative variations” of a listed polypeptide sequence of thepresent invention include substitutions of a small percentage, typicallyless than 5%, more typically less than 2% or 1%, of the amino acids ofthe polypeptide sequence, with an amino acid of the same conservativesubstitution group. Finally, the addition of sequences which do notalter the encoded activity of a nucleic acid molecule, such as theaddition of a non-functional or tagging sequence (introns in the nucleicacid, poly His or similar sequences in the encoded polypeptide, etc.),is a conservative variation of the basic nucleic acid or polypeptide.

Conservative substitution tables providing functionally similar aminoacids are well known in the art, where one amino acid residue issubstituted for another amino acid residue having similar chemicalproperties (e.g., aromatic side chains or positively charged sidechains), and therefore does not substantially change the functionalproperties of the polypeptide molecule. The following sets forth examplegroups that contain natural amino acids of like chemical properties,where substitutions within a group is a “conservative substitution”.

TABLE 1 Conservative amino acid substitutions Nonpolar and/or Polar,Positively Negatively Aliphatic Uncharged Aromatic Charged Charged SideSide Side Side Side Chains Chains Chains Chains Chains Glycine SerinePhenylalanine Lysine Aspartate Alanine Threonine Tyrosine ArginineGlutamate Valine Cysteine Tryptophan Histidine Leucine MethionineIsoleucine Asparagine Proline Glutamine

Nucleic Acid Hybridization

Comparative hybridization can be used to identify nucleic acids of theinvention, including conservative variations of nucleic acids of theinvention. In addition, target nucleic acids which hybridize to anucleic acid of the invention under high, ultra-high and ultra-ultrahigh stringency conditions, where the nucleic acids encode mutantscorresponding to those noted in Tables 3-10 and FIGS. 7-9 or otherlisted polymerases, are a feature of the invention. Examples of suchnucleic acids include those with one or a few silent or conservativenucleic acid substitutions as compared to a given nucleic acid sequenceencoding a polymerase of Tables 3-10 and FIGS. 7-9 (or other exemplifiedpolymerase), where any conservative substitutions are for residues otherthan those noted in Tables 3-10 and FIGS. 7-9 or elsewhere as beingrelevant to a feature of interest (increased stability or readlength,improved performance with large analogs, etc.).

A test nucleic acid is said to specifically hybridize to a probe nucleicacid when it hybridizes at least 50% as well to the probe as to theperfectly matched complementary target, i.e., with a signal to noiseratio at least half as high as hybridization of the probe to the targetunder conditions in which the perfectly matched probe binds to theperfectly matched complementary target with a signal to noise ratio thatis at least about 5×-10× as high as that observed for hybridization toany of the unmatched target nucleic acids.

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part I chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays,” (Elsevier, New York), aswell as in Current Protocols in Molecular Biology, Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (supplemented through 2012); Hames andHiggins (1995) Gene Probes 1 IRL Press at Oxford University Press,Oxford, England, (Hames and Higgins 1) and Hames and Higgins (1995) GeneProbes 2 IRL Press at Oxford University Press, Oxford, England (Hamesand Higgins 2) provide details on the synthesis, labeling, detection andquantification of DNA and RNA, including oligonucleotides.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formalin with1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of stringent wash conditions is a 0.2×SSC wash at65° C. for 15 minutes (see, Sambrook, supra for a description of SSCbuffer). Often the high stringency wash is preceded by a low stringencywash to remove background probe signal. An example low stringency washis 2×SSC at 40° C. for 15 minutes. In general, a signal to noise ratioof 5× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization.

“Stringent hybridization wash conditions” in the context of nucleic acidhybridization experiments such as Southern and northern hybridizationsare sequence dependent, and are different under different environmentalparameters. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993), supra. and in Hames and Higgins, 1 and 2.Stringent hybridization and wash conditions can easily be determinedempirically for any test nucleic acid. For example, in determiningstringent hybridization and wash conditions, the hybridization and washconditions are gradually increased (e.g., by increasing temperature,decreasing salt concentration, increasing detergent concentration and/orincreasing the concentration of organic solvents such as formalin in thehybridization or wash), until a selected set of criteria are met. Forexample, in highly stringent hybridization and wash conditions, thehybridization and wash conditions are gradually increased until a probebinds to a perfectly matched complementary target with a signal to noiseratio that is at least 5× as high as that observed for hybridization ofthe probe to an unmatched target.

“Very stringent” conditions are selected to be equal to the thermalmelting point (T_(m)) for a particular probe. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetest sequence hybridizes to a perfectly matched probe. For the purposesof the present invention, generally, “highly stringent” hybridizationand wash conditions are selected to be about 5° C. lower than the T_(m)for the specific sequence at a defined ionic strength and pH.

“Ultra high-stringency” hybridization and wash conditions are those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10× ashigh as that observed for hybridization to any of the unmatched targetnucleic acids. A target nucleic acid which hybridizes to a probe undersuch conditions, with a signal to noise ratio of at least ½ that of theperfectly matched complementary target nucleic acid is said to bind tothe probe under ultra-high stringency conditions.

Similarly, even higher levels of stringency can be determined bygradually increasing the hybridization and/or wash conditions of therelevant hybridization assay. For example, those in which the stringencyof hybridization and wash conditions are increased until the signal tonoise ratio for binding of the probe to the perfectly matchedcomplementary target nucleic acid is at least 10×, 20×, 50×, 100×, or500× or more as high as that observed for hybridization to any of theunmatched target nucleic acids. A target nucleic acid which hybridizesto a probe under such conditions, with a signal to noise ratio of atleast ½ that of the perfectly matched complementary target nucleic acidis said to bind to the probe under ultra-ultra-high stringencyconditions.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, e.g., when a copyof a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

Unique Subsequences

In some aspects, the invention provides a nucleic acid that comprises aunique subsequence in a nucleic acid that encodes a polymerase of Tables3-10 and FIGS. 7-9 or others described herein. The unique subsequencemay be unique as compared to a nucleic acid corresponding to, e.g., awild type Φ29-type polymerase. Alignment can be performed using, e.g.,BLAST set to default parameters. Any unique subsequence is useful, e.g.,as a probe to identify the nucleic acids of the invention.

Similarly, the invention includes a polypeptide which comprises a uniquesubsequence in a polymerase of Tables 3-10 and FIGS. 7-9 or otherwisedetailed herein. Here, the unique subsequence is unique as compared to,e.g., a wild type Φ29-type polymerase or previously characterizedmutation thereof.

The invention also provides for target nucleic acids which hybridizeunder stringent conditions to a unique coding oligonucleotide whichencodes a unique subsequence in a polypeptide selected from the modifiedpolymerase sequences of the invention, wherein the unique subsequence isunique as compared to a polypeptide corresponding to a wild typeΦ29-type polymerase. Unique sequences are determined as noted above.

Sequence Comparison, Identity, and Homology

The terms “identical” or “percent identity,” in the context of two ormore nucleic acid or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the sequence comparison algorithms described below (or otheralgorithms available to persons of skill) or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides (e.g., DNAs encoding a polymerase, or the aminoacid sequence of a polymerase) refers to two or more sequences orsubsequences that have at least about 60%, about 80%, about 90%, about95%, about 98%, about 99% or more nucleotide or amino acid residueidentity, when compared and aligned for maximum correspondence, asmeasured using a sequence comparison algorithm or by visual inspection.Such “substantially identical” sequences are typically considered to be“homologous,” without reference to actual ancestry. Preferably, the“substantial identity” exists over a region of the sequences that is atleast about 50 residues in length, more preferably over a region of atleast about 100 residues, and most preferably, the sequences aresubstantially identical over at least about 150 residues, or over thefull length of the two sequences to be compared.

Proteins and/or protein sequences are “homologous” when they arederived, naturally or artificially, from a common ancestral protein orprotein sequence. Similarly, nucleic acids and/or nucleic acid sequencesare homologous when they are derived, naturally or artificially, from acommon ancestral nucleic acid or nucleic acid sequence. Homology isgenerally inferred from sequence similarity between two or more nucleicacids or proteins (or sequences thereof). The precise percentage ofsimilarity between sequences that is useful in establishing homologyvaries with the nucleic acid and protein at issue, but as little as 25%sequence similarity over 50, 100, 150 or more residues is routinely usedto establish homology. Higher levels of sequence similarity, e.g., 30%,40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, or 99% or moreidentity, can also be used to establish homology. Methods fordetermining sequence similarity percentages (e.g., BLASTP and BLASTNusing default parameters) are described herein and are generallyavailable.

For sequence comparison and homology determination, typically onesequence acts as a reference sequence to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyCurrent Protocols in Molecular Biology, Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., supplemented through 2012).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993) Proc. Nat'l. Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

For reference, the amino acid sequence of a wild-type Φ29 polymerase ispresented in Table 2, along with the sequences of several otherwild-type Φ29-type polymerases.

TABLE 2 Amino acid sequence of exemplary wild-type Φ29-type polymerases.Φ29 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHSEYKIG SEQ ID NO: 1NSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPK PVQVPGGVVLVDDTFTIK M2YMSRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLD SEQ ID NO: 2EFMQWVMEIQADLYFHNLKFDGAFIVNWLEQHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHTERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRKAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGVEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKDFIDKWTYVKTHEEGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLKDDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYVKEVDGKLKECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFAVGFSSMGKPKPVQ VNGGVVLVDSVFTIK B103MPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLD SEQ ID NO: 3EFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHAERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRRAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGAEPVELYLTNVDLELIQEHYEMYNVEYIDGFKFREKTGLFKEFIDKWTYVKTHEKGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYAKEVDGKLIECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFRVGFSSTGKPKPVQVN GGVVLVDSVFTIK GA-1MARSVYVCDFETTTDPEDCRLWAWGWMDIYNTDKWSYGEDI SEQ ID NO: 4DSFMEWALNSNSDIYFHNLKFDGSFILPWWLRNGYVHTEEDRTNTPKEFTTTISGMGQWYAVDVCINTRGKNKNHVVFYDSLKKLPFKVEQIAKGFGLPVLKGDIDYKKYRPVGYVMDDNEIEYLKHDLLIVALALRSMFDNDFTSMTVGSDALNTYKEMLGVKQWEKYFPVLSLKVNSEIRKAYKGGFTWVNPKYQGETVYGGMVFDVNSMYPAMMKNKLLPYGEPVMFKGEYKKNVEYPLYIQQVRCFFELKKDKIPCIQIKGNARFGQNEYLSTSGDEYVDLYVTNVDWELIKKHYDIFEEEFIGGFMFKGFIGFFDEYIDRFMEIKNSPDSSAEQSLQAKLMLNSLYGKFATNPDITGKVPYLDENGVLKFRKGELKERDPVYTPMGCFITAYARENILSNAQKLYPRFIYADTDSIHVEGLGEVDAIKDVIDPKKLGYWDHEATFQRARYVRQKTYFIETTWKENDKGKLVVCEPQDATKVKPKIACAGMSDAIKERIRFNEF KIGYSTHGSLKPKNVLGGVVLMDYPFAIKAV-1 MVRQSTIASPARGGVRRSHKKVPSFCADFETTTDEDDCRVWS SEQ ID NO: 5WGIIQVGKLQNYVDGISLDGFMSHISERASHIYFHNLAFDGTFILDWLLKHGYRWTKENPGVKEFTSLISRMGKYYSITVVFETGFRVEFRDSFKKLPMSVSAIAKAFNLHDQKLEIDYEKPRPIGYIPTEQEKRYQRNDVAIVAQALEVQFAEKMTKLTAGSDSLATYKKMTGKLFIRRFPILSPEIDTEIRKAYRGGFTYADPRYAKKLNGKGSVYDVNSLYPSVMRTALLPYGEPIYSEGAPRTNRPLYIASITFTAKLKPNHIPCIQIKKNLSFNPTQYLEEVKEPTTVVATNIDIELWKKHYDFKIYSWNGTFEFRGSHGFFDTYVDHFMEIKKNSTGGLRQIAKLHLNSLYGKFATNPDITGKHPTLKDNRVSLVMNEPETRDPVYTPMGVFITAYARKKTISAAQDNYETFAYADTDSLHLIGPTTPPDSLWVDPVELGAWKHESSFTKSVYIRAKQYAEEIGGKLDVHIAGMPRNVAATLTLEDMLHGGTWNGKLIPVRVPGGTVLKDT TFTLKID CP-1MTCYYAGDFETTTNEEETEVWLSCFAKVIDYDKLDTFKVNTS SEQ ID NO: 6LEDFLKSLYLDLDKTYTETGEDEFIIFFHNLKPDGSFLLSFFLNNDIECTYFINDMGVWYSITLEFPDFTLTFRDSLKILNFSIATMAGLFKMPIAKGTTPLLKHKPEVIKPEWIDYIHVDVAILARGIFAMYYEENFTKYTSASEALTEFKRIFRKSKRKFRDFFPILDEKVDDFCRKHIVGAGRLPTLKHRGRTLNQLIDIYDINSMYPATMLQNALPIGIPKRYKGKPKEIKEDHYYIYHIKADFDLKRGYLPTIQIKKKLDALRIGVRTSDYVTTSKNEVIDLYLTNFDLDLFLKHYDATIMYVETLEFQTESDLFDDYITTYRYKKENAQSPAEKQKAKIMLNSLYGKFGAKIISVKKLAYLDDKGILRFKNDDEEEVQPVYAPVALFVTSIARHFIISNAQENYDNFLYADTDSLHLFHSDSLVLDIDPSEFGKWAHEGRAVKAKYLRSKLYIEELIQEDGTTHLDVKGAGMTPEIKEKITFENFVIGATFEGKRASKQIKGGTLIYETTFKIRETDYLV

Exemplary Mutation Combinations

A list of exemplary polymerase mutation combinations, and optionalcorresponding exogenous or heterologous features at the C-terminalregion of the polymerase, is provided in Tables 3 and 4. Positions ofamino acid substitutions are identified relative to a wild-type Φ29 DNApolymerase (SEQ ID NO:1) for the recombinant polymerases in Table 3 andrelative to a wild-type M2Y DNA polymerase (SEQ ID NO:2) for therecombinant polymerases in Table 4. Polymerases of the invention(including those provided in Tables 3 and 4) can include any exogenousor heterologous feature (or combination of such features), e.g., at theN- and/or C-terminal region. For example, it will be understood thatpolymerase mutants in Tables 3 and 4 that do not include, e.g., aC-terminal biotinylation site can be modified to include a biotinylationsite at the C-terminal region, alone or in combination with any of theexogenous or heterologous features described herein. Similarly, some orall of the exogenous features listed in Tables 3 and 4 can be omitted,or substituted or combined with any of the other exogenous featuresdescribed herein, and still result in a polymerase of the invention. Aswill be appreciated, the numbering of amino acid residues is withrespect to a particular reference polymerase, such as the wild-typesequence of the Φ29 polymerase (SEQ ID NO:1) or M2Y polymerase (SEQ IDNO:2); actual position of a mutation within a molecule of the inventionmay vary based upon the nature of the various modifications that theenzyme includes relative to the wild type Φ29 enzyme, e.g., deletionsand/or additions to the molecule, either at the termini or within themolecule itself.

TABLE 3Exemplary mutations introduced into a Φ29 DNA polymerase. Positionsare identified relative to SEQ ID NO: 1. C-terminal region Mutationsfeature(s) K131E L142K Y224K D235E E239G V250A L253H His10E375Y A437G A484E E508R D510K K512Y E515Q GGGSGGGSGGGS BtagV7K131E Y224K E239G V250I L253A E375Y A437G His10C455A A484E D510K K512Y E515Q GGGSGGGSGGGS BtagV7C11A C106S K131E Y224K E239G L253H C290F His10K337C E375Y A437G C448V A484E D510K K512Y GGGSGGGSGGGS E515Q BtagV7K131E A134S Y148I Y224K D235E E239G L253H His10E375Y A437G A484E D510K K512Y E515Q GGGSGGGSGGGS BtagV7K131E Y148I Y224K E239G V250I L253A E375Y His10A437G A484E D510K K512Y E515Q D570E GGGSGGGSGGGS BtagV7Q99W K131E Y148I Y224K E239G V250I L253A His10E375Y A437G A484E D510K K512Y E515Q GGGSGGGSGGGS BtagV7K131E Y224K E239G V250I L253A A256S E375Y His10A437G C455A A484E D510K K512Y E515Q F526L GGGSGGGSGGGS D570E BtagV7Q991 K131E A134S Y224K E239G V250I L253A His10E375Y A437N A484E E508R D510K K512Y E515Q GGGSGGGSGGGS D570E BtagV7Q99I K131E A134S Y224K E239G V250I L253A His10R306Q E375Y A437N A484E E508R D510K K512Y GGGSGGGSGGGS E515Q D570EBtagV7 K131E Y224K E239G L253H E375Y M429A A437G His10C455A A484E D510K K512Y E515Q GGGSGGGSGGGS BtagV7K131E Y148I Y224K E239G V250I L253A E375Y His10A437G A484E D510K K512Y E515Q GGGSGGGSGGGSK131E A134S Y224K E239G V250I L253A E375Y His10A437N A484E E508R D510K K512Y E515Q D570E GGGSGGGSGGGS BtagV7K131E Y148I Y224K E239G V250I L253A E375Y His10A437G A484E H485Q D510K K512Y E515Q GGGSGGGSGGGS BtagV7K131E Y148I Y224K E239G V250I L253A E375Y His10M429A A437G A484E D510K K512Y E515Q GGGSGGGSGGGS BtagV7V141K L142K Y224K E239G V250I L253A A256S His10R306Q R308L K311E E375Y A437G T441I A484E GGGSGGGSGGGSS487A E508R D510K K512Y E515Q BtagV7Y224K E239G V250I L253A A256S E375Y A437G His10A484E S487A D510K K512Y E515Q GGGSGGGSGGGS BtagV7V141K L142K Y224K E239G V250I L253A A256S His10E375Y A437G T441I A484E S487A E508R D510K GGGSGGGSGGGS K512Y E515Q D570EBtagV7 A134S Y224K E239G V250I L253A A256S E375Y His10A437G A484E S487A D510K K512Y E515Q GGGSGGGSGGGS BtagV7

TABLE 4 Exemplary mutations introduced into an M2Y DNA polymerase.Positions are identified relative to SEQ ID NO: 2. C-terminal regionMutations feature(s) K128E Y145I E236G V247I L250A S253A E372Y His10A434G A481E D507K K509Y E512Q GGGSGGGSGGGS BtagV7C1035 K128E K132Q R172E E236G V247I L250A His10S253A E372Y A434G A481E E505R D507K K509Y GGGSGGGSGGGS E512Q BtagV7

The amino acid sequences of exemplary recombinant Φ29 and M2Ypolymerases harboring the exemplary mutation combinations of Tables 3and 4 are provided in Tables 5 and 6. Table 5 includes the polymeraseportion of the molecule as well as the one or more exogenous features atthe C-terminal region of the polymerase, while Table 6 includes theamino acid sequence of the polymerase portion only.

TABLE 5 Amino acid sequences of exemplary recombinant Φ29 and M2Ypolymerases including C-terminal exogenous features. Amino acidpositions are identified relative to SEQ ID NO: 1 for recombinantΦ29 polymerases (denoted by ″Phi29″) or relative to SEQ ID NO: 2for recombinant M2Y polymerases (denoted by ″M2″). SEQ ID NOAmino Acid Sequence  7 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K131E_L142K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIY224K_D235E_E239G_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICV250A_L253H_E375Y_ LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVKKA437G_A484E_E508R_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKD510K_K512Y_E515Q. QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDHis10.GGGSGGGSGGGS. KEVRKAYRGGFTWLNERFKGKEIGEGMVFDANSHY BtagV7PAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKG YLVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHH HHHHGGGSGGGSGGGSGLNDFFEAQKIEWHE  8MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.K131E_Y224K_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI E239G_V250I_L253A_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC E375Y_A437G_C455A_LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLK A484E_D510K_K512Y_GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK E515Q.His10.QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD GGGSGGGSGGGS.BtagV7KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYADTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE  9MKHMPRKMYSADFETTTKVEDCRVWAYGYMNIED Phi29.C11A_C106S_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI K131E_Y224K_E239G_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIS L253H_C290F_K337C_LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLK E375Y_A437G_C448V_GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK A484E_D510K_K512Y_QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD E515Q.His10.KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDVNSHY GGGSGGGSGGGS.BtagV7PAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRFEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMCEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQAVYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKG YLVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHH HHHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 10MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.K131E_A134S_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI Y148I_Y224K_D235E_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC E239G_L253H_E375Y_LGYKGKRKIHTVIYDSLKKLPFPVEKISKDFKLTVLK A437G_A484E_D510K_GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK K512Y_E515Q.His10.QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD GGGSGGGSGGGS.BtagV7KEVRKAYRGGFTWLNERFKGKEIGEGMVFDVNSHYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKG YLVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHH HHHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 11MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.K131E_Y1481_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI Y224K_E239G_V250I_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC L253A_E375Y_A437G_LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLK A484E_D510K_K512Y_GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK E515Q_D570E.His10.QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD GGGSGGGSGGGS.BtagV7KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 12MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.Q99W_K131E_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI Y148I_Y224K_E239G_INWLERNGFKWSADGLPNTYNTIISRMGWWYMIDIC V250I_L253A_E375Y_LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLK A437G_A484E_D510K_GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK K512Y_E515Q.His10.QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD GGGSGGGSGGGS.BtagV7KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 13MSRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNY M2.K128E_Y1451_E236G_KIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWL V247I_L250A_S253A_EQHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYK E372Y_A434G_A481E_GKRKLHTVIYDSLKKLPFPVEKIAKDFQLPLLKGDIDI D507K_K509Y_E512Q.HTERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDR His10.GGGSGGGSGGGS.MTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRK BtagV7AYRGGFTWLNDKYKGKEIGEGMVFDINSAYPAQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGVEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKDFIDKWTYVKTHEYGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLK DDGSLGFRVGDEEYKDPVYTPMGVFITAWGRFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYVKEVKGYLKQCSPDEATTTKFSVKCAGMTDTIKKKVTFDNFAVGFSSM GKPKPVQVNGGVVLVDSVFTIKGHHHHHHHHHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 14 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K131E_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICA256S_E375Y_A437G_ LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLKC455A_A484E_D510K_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKK512Y_E515Q_F526L_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD D570E.His10.KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP GGGSGGGSGGGS.BtagV7SQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYADTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKLSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 15MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.Q99I_K131E_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI A134S_Y224K_E239G_INWLERNGFKWSADGLPNTYNTIISRMGIWYMIDICL V250I_L253A_E375Y_GYKGKRKIHTVIYDSLKKLPFPVEKISKDFKLTVLKG A437N_A484E_E508R_DIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQ D510K_K512Y_E515Q_GLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDK D570E.His10.EVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPA GGGSGGGSGGGS.BtagV7QMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWNRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGYL VQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIKGHHHHHHHH HHGGGSGGGSGGGSGLNDFFEAQKIEWHE 16MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.Q99I_K131E_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI A134S_Y224K_E239G_INWLERNGFKWSADGLPNTYNTIISRMGIWYMIDICL V250I_L253A_R306Q_GYKGKRKIHTVIYDSLKKLPFPVEKISKDFKLTVLKG E375Y_A437N_A484E_DIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQ E508R_D510K_K512Y_GLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDK E515Q_D570E.His10.EVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPA GGGSGGGSGGGS.BtagV7QMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKQSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWNRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 17MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.K131E_Y224K_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI E239G_L253H_E375Y_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC M429A_A437G_C455A_LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLK A484E_D510K_K512Y_GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK E515Q.His10.QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD GGGSGGGSGGGS.BtagV7KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDVNSHYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPAGVFITAWGRYTTITAAQACYDRIIYADTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKG YLVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHH HHHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 18MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.K131E_Y1481_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI Y224K_E239G_V250I_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC L253A_E375Y_A437G_LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLK A484E_D510K_K512Y_GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK E515Q.His10.QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD GGGSGGGSGGGSKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHH HHHGGGSGGGSGGGS 19MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.K131E_A134S_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI Y224K_E239G_V250I_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC L253A_E375Y_A437N_LGYKGKRKIHTVIYDSLKKLPFPVEKISKDFKLTVLK A484E_E508R_D510K_GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK K512Y_E515Q_D570E.QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD His10.GGGSGGGSGGGS.KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP BtagV7AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWNRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 20MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.K131E_Y1481_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI Y224K_E239G_V250I_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC L253A_E375Y_A437G_LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLK A484E_H485Q_D510K_GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK K512Y_E515Q.His10.QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD GGGSGGGSGGGS.BtagV7KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEQESTFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 21MSRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNY M2.C103S_K128E_K132Q_KIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWL R172E E236G V247I_EQHGFKWSNEGLPNTYNTIISKMGQWYMIDISFGYK L250A_S253A_E372Y_GKRKLHTVIYDSLKKLPFPVEKIAQDFQLPLLKGDID A434G A481E E505R_YHTERPVGHEITPEEYEYIKNDIEIIAEALDIQFKQGLD D507K_K509Y E512Q.RMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIR His10.GGGSGGGSGGGS.KAYRGGFTWLNDKYKGKEIGEGMVFDINSAYPAQM BtagV7YSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGVEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKDFIDKWTYVKTHEYGAKKQLAKLMLNSLYGKFASNPDVTGKVPY LKDDGSLGFRVGDEEYKDPVYTPMGVFITAWGRFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYVKRVKGYLKQCSPDEATTTKFSVKCAGMTDTIKKKVTFDNFAVGFSS MGKPKPVQVNGGVVLVDSVFTIKGHHHHHHHHHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 22 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K131E_Y1481_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIY224K_E239G_V250I_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICL253A_E375Y_M429A_ LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLKA437G_A484E_D510K_ GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKK512Y_E515Q.His10. QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDGGGSGGGSGGGS.BtagV7 KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPAGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 23MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.V141K_L142K_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI Y224K E239G_V250I_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC L253A_A256S_R306Q_LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTKKK R308L_K311E_E375Y_GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK A437G_T441I_A484E_QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD S487A_E508R_D510K_KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP K512Y_E515Q.His10.SQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF GGGSGGGSGGGS.BtagV7ELKEGYIPTIQIKQSLFYEGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTIITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHEATFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 24MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.Y224K_E239G_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI V250I_L253A_A256S_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC E375Y_A437G_A484E_LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLK S487A_D510K_K512Y_GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK E515Q.His10.QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD GGGSGGGSGGGS.BtagV7KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPSQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHEATFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 25MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.V141K_L142K_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI Y224K_E239G_V250I_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC L253A_A256S_E375Y_LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTKKK A437G_T441I_A484E_GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK S487A_E508R_D510K_QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD K512Y_E515Q_D570E.KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP His10.GGGSGGGSGGGS.SQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF BtagV7ELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTIITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHEATFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE 26MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.A134S_Y224K_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI E239G_V250I_L253A_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC A256S_E375Y_A437G_LGYKGKRKIHTVIYDSLKKLPFPVKKISKDFKLTVLK A484E_S487A_D510K_GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK K512Y_E515Q.His10.QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD GGGSGGGSGGGS.BtagV7KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPSQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHEATFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE

TABLE 6Amino acid sequences of exemplary recombinant Φ29 and M2Y polymerases.Amino acid positions are identified relative to SEQ ID NO: 1 forrecombinant Φ29 polymerases (denoted by ″Phi29″) or relative to SEQ IDNO: 2 for recombinant M2Y polymerases (denoted by ″M2″). SEQ ID NOAmino Acid Sequence 27 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K131E_L142K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIY224K_D235E_E239G_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICV250A_L253H_E375Y_ LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVKKA437G_A484E_E508R_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKD510K_K512Y_E515Q QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNERFKGKEIGEGMVFDANSHYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKG YLVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 28 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K131E_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICE375Y_A437G_C455A_ LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLKA484E_D510K_K512Y_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK E515QQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYADTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 29 MKHMPRKMYSADFETTTKVEDCRVWAYGYMNIEDPhi29.C11A_C106S_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIK131E_Y224K_E239G_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDISL253H_C290F_K337C_ LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLKE375Y_A437G_C448V_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKA484E_D510K_K512Y_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD E515QKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDVNSHYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRFEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMCEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQAVYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKG YLVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 30 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K131E_A134S_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIY148I_Y224K_D235E_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICE239G_L253H_E375Y_ LGYKGKRKIHTVIYDSLKKLPFPVEKISKDFKLTVLKA437G_A484E_D510K_ GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK K512Y_E515QQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNERFKGKEIGEGMVFDVNSHYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKG YLVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 31 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K131E_Y148I_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIY224K_E239G_V250I_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICL253A_E375Y_A437G_ LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLKA484E_D510K_K512Y_ GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK E515Q_D570EQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIK 32 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.Q99W_K131E_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIY148I_Y224K_E239G_ INWLERNGFKWSADGLPNTYNTIISRMGWWYMIDICV250I_L253A_E375Y_ LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLKA437G_A484E_D510K_ GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK K512Y_E515QQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 33 MSRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYM2.K128E_Y1451_E236G_ KIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLV247I_L250A_S253A_ EQHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKE372Y_A434G_A481E_ GKRKLHTVIYDSLKKLPFPVEKIAKDFQLPLLKGDIDID507K_K509Y_E512Q HTERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRKAYRGGFTWLNDKYKGKEIGEGMVFDINSAYPAQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGVEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKDFIDKWTYVKTHEYGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLK DDGSLGFRVGDEEYKDPVYTPMGVFITAWGRFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYVKEVKGYLKQCSPDEATTTKFSVKCAGMTDTIKKKVTFDNFAVGFSSM GKPKPVQVNGGVVLVDSVFTIK 34MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.K131E_Y224K_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI E239G_V250I_L253A_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC A256S_E375Y_A437G_LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLK C455A_A484E_D510K_GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK K512Y_E515Q_F526L_QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD D570EKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPSQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYADTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKLSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIK 35 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.Q99I_K131E_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIA134S_Y224K_E239G_ INWLERNGFKWSADGLPNTYNTIISRMGIWYMIDICLV250I_L253A_E375Y_ GYKGKRKIHTVIYDSLKKLPFPVEKISKDFKLTVLKGA437N_A484E_E508R_ DIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQD510K_K512Y_E515Q_ GLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDK D570EEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWNRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGYL VQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIK 36 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.Q99I_K131E_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIA134S_Y224K_E239G_ INWLERNGFKWSADGLPNTYNTIISRMGIWYMIDICLV250I_L253A_R306Q_ GYKGKRKIHTVIYDSLKKLPFPVEKISKDFKLTVLKGE375Y_A437N A484E_ DIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQE508R_D510K_K512Y_ GLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDK E515Q_D570EEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKQSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWNRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIK 37 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K131E_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_L253H_E375Y_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICM429A_A437G_C455A_ LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLKA484E_D510K_K512Y_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK E515QQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDVNSHYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPAGVFITAWGRYTTITAAQACYDRIIYADTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKG YLVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 38 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K131E_Y148I_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIY224K_E239G_V250I_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICL253A_E375Y_A437G_ LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLKA484E_D510K_K512Y_ GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK E515QQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 39 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K131E_A134S_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIY224K_E239G_V250I_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICL253A_E375Y_A437N_ LGYKGKRKIHTVIYDSLKKLPFPVEKISKDFKLTVLKA484E_E508R_D510K_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKK512Y_E515Q_D570E QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWNRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIK 40 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K131E_Y1481_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIY224K_E239G_V250I_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICL253A_E375Y_A437G_ LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLKA484E_H485Q_D510K_ GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK K512Y_E515QQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEQESTFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 41 MSRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYM2.C103S_K128E_K132Q_ KIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLR172E_E236G_V247I_ EQHGFKWSNEGLPNTYNTIISKMGQWYMIDISFGYKL250A S253A_E372Y_ GKRKLHTVIYDSLKKLPFPVEKIAQDFQLPLLKGDIDA434G_A481E_E505R_ YHTERPVGHEITPEEYEYIKNDIEIIAEALDIQFKQGLDD507K_K509Y_E512Q RMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRKAYRGGFTWLNDKYKGKEIGEGMVFDINSAYPAQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGVEPVELYLTNVDLELIQEHYELYNVEYIDGFICFREKTGLFKDFIDKWTYVKTHEYGAKKQLAKLMLNSLYGKFASNPDVTGKVPY LKDDGSLGFRVGDEEYKDPVYTPMGVFITAWGRFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYVKRVKGYLKQCSPDEATTTKFSVKCAGMTDTIKKKVTFDNFAVGFSS MGKPKPVQVNGGVVLVDSVFTIK 42MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.K131E_Y148I_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI Y224K_E239G_V250I_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC L253A_E375Y_M429A_LGYKGKRKIHTVIYDSLKKLPFPVEKIAKDFKLTVLK A437G_A484E_D510K_GDIDIHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK K512Y_E515QQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPAGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 43 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.V141K_L142K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIY224K_E239G_V250I_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICL253A_A256S_R306Q_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTKKKR308L_K311E_E375Y_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKA437G_T441I_A484E_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDS487A_E508R_D510K_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP K512Y_E515QSQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKQSLFYEGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTIITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHEATFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 44 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.Y224K_E239G_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIV250I_L253A_A256S_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICE375Y_A437G_A484E_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKS487A_D510K_K512Y_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK E515QQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPSQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHEATFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK 45 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.V141K_L142K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIY224K_E239G_V250I_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICL253A_A256S_E375Y_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTKKKA437G_T441I_A484E_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKS487A_E508R_D510K_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDK512Y_E515Q_D570E KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPSQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTIITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHEATFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIK 46 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.A134S_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICA256S_E375Y_A437G_ LGYKGKRKIHTVIYDSLKKLPFPVKKISKDFKLTVLKA484E_S487A_D510K_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK K512Y_E515QQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPSQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHEATFKRAKYLRQKTYIQDIYMKEVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK

A list of additional exemplary polymerase mutation combinations, andoptional corresponding exogenous or heterologous features at theC-terminal region of the polymerase, is provided in Tables 7 and 8.These combinations can enhance performance of the polymerase with largeanalogs (e.g., protein shield analogs, other shielded analogs, or otheranalogs with a molecular weight of at least 10,000), e.g., in singlemolecule sequencing. Positions of amino acid substitutions areidentified relative to a wild-type Φ29 DNA polymerase (SEQ ID NO:1) forthe recombinant polymerases in Table 7 and relative to a wild-type M2YDNA polymerase (SEQ ID NO:2) for the recombinant polymerase in Table 8.Polymerases of the invention (including those provided in Tables 7 and8) can include any exogenous or heterologous feature (or combination ofsuch features), e.g., at the N- and/or C-terminal region. For example,some or all of the exogenous features listed in Tables 7 and 8 can beomitted, or substituted or combined with any of the other exogenousfeatures described herein, and still result in a polymerase of theinvention. As will be appreciated, the numbering of amino acid residuesis with respect to a particular reference polymerase, such as thewild-type sequence of the Φ29 polymerase (SEQ ID NO:1) or M2Y polymerase(SEQ ID NO:2); actual position of a mutation within a molecule of theinvention may vary based upon the nature of the various modificationsthat the enzyme includes relative to the wild type Φ29 enzyme, e.g.,deletions and/or additions to the molecule, either at the termini orwithin the molecule itself.

TABLE 7Exemplary mutations introduced into a Φ29 DNA polymerase. Positions areidentified relative to SEQ ID NO: 1. C-terminal region Mutationsfeature(s) Y224K E239G V250I L253A E375Y A437G A484E His10E508R D510K K512Y E515Q D570M GGGSGGGSGGGS BtagV7A134S Y224K E239G V250I L253A E375Y A437G His10A484E S487A E508R D510K K512Y E515Q I524S GGGSGGGSGGGS F526L D570EBtagV7 L142K Y224K E239G V250I L253A E375Y A437G His10D476H A484E E508R D510K K512Y L513K E515Q GGGSGGGSGGGS D570E BtagV7GGGSGGGSGGGS BtagV7 L142K Y224K E239G V250I L253A R306Q R308L His10E375Y A437G D476H A484E E508R D510K K512Y GGGSGGGSGGGS L513K E515Q D570EBtagV7 GGGSGGGSGGGS BtagV7 L142K Y224K E239G V250I L253A R306Q R308LHis10 E375Y A437G E466K D476H A484E E508R D510K GGGSGGGSGGGSK512Y L513K E515Q D570E BtagV7 GGGSGGGSGGGS BtagV7L142K Y224K E239G V250I L253A R306Q R308L His10E375Y A437G E466K D476H A484E E508R D510K GGGSGGGSGGGS K512Y E515Q D570EBtagV7 GGGSGGGSGGGS BtagV7 L142K Y224K E239G V250I L253A E375Y A437GHis10 E466K D476H A484E E508R D510K K512Y E515Q GGGSGGGSGGGS D570EBtagV7 GGGSGGGSGGGS BtagV7 L142K Y224K E239G V250I L253A E375Y M429AHis10 A437G A484E E508R D510K K512Y E515Q D570E GGGSGGGSGGGS BtagV7GGGSGGGSGGGS BtagV7 A134S L142K Y224K E239G V250I L253A E375Y His10A437G A484E S487A E508R D510K K512Y E515Q GGGSGGGSGGGS I524S F526L D570EBtagV7 L142K Y224K D235E E239G V250A L253H E375Y His10A437G A484E E508R D510K K512Y E515Q D570E GGGSGGGSGGGS BtagV7Y224K E239G V250I L253A E375Y A437G A484E His10 E508R D510K K512Y E515QGGGSGGGSGGGS BtagV7 V141K L142K Y224K E239G V250I L253A E375Y His10A437G A484E E508R D510K K512Y E515Q GGGSGGGSGGGS BtagV7Y224K E239G V250I L253A E375Y A437G A484E His10S487A E508R D510K K512Y E515Q I524S F526L GGGSGGGSGGGS D570M BtagV7K135Q L142K Y224K E239G V250I L253A R306Q His10R308L E375Y A437G E466K D476H A484E E508R GGGSGGGSGGGSD510R K512Y E515Q D570S T571V BtagV7 GGGSGGGSGGGS BtagV7K131E K135Q L142K Y224K E239G V250I L253A His10E375Y A437G A484E E508R D510R K512Y E515Q GGGSGGGSGGGSD523R P558A D570S T571V BtagV7 GGGSGGGSGGGS BtagV7A49E C106S K114R K131E K135Q L142K Y224K His10E239G V250I L253A Y369E E375Y A437G D476H GGGSGGGSGGGSA484E E508R D510K K512Y E515Q D523R D570S BtagV7 T571V GGGSGGGSGGGSBtagV7 K135Q K138Q L142K Y224K E239G V250I L253A His10R306Q R308L E375Y A437G E466K D476H A484E GGGSGGGSGGGSE508R D510K K512Y E515Q I524T P558A D570S BtagV7 T571V GGGSGGGSGGGSBtagV7 K135Q K138Q L142K Y224K E239G V250I L253A His10R306Q R308L E375Y A437G E466K V475I D476H GGGSGGGSGGGSA484E E508R D510K K512Y E515Q I524T P558A BtagV7 D570S T571VGGGSGGGSGGGS BtagV7 K135Q K138Q L142K Y224K E239G V250I L253A His10R306Q R308L T368S E375Y A437G E466K D476H GGGSGGGSGGGSA484E E508R D510K K512Y E515Q I524T P558A BtagV7 D570S T571VGGGSGGGSGGGS BtagV7 K135Q K138Q L142K Y224K E239G V250I L253A His10R306Q R308L T368S E375Y A437G E466K V475I GGGSGGGSGGGSD476H A484E E508R D510K K512Y E515Q P558A BtagV7 D570S T571VGGGSGGGSGGGS BtagV7 L44A K135Q K138Q L142K Y224K E239G V250I His10L253A R306Q R308L E375Y A437G E466K D476H GGGSGGGSGGGSA484E S487A E508R D510K K512Y E515Q P558A BtagV7 D570S T571VGGGSGGGSGGGS BtagV7 K135Q L142K Y224K E239G V250I L253A R306Q His10R308L T368S E375Y A437G E466K D476H A484E GGGSGGGSGGGSE508R D510R K512Y E515Q P558A D570S T571V BtagV7 GGGSGGGSGGGS BtagV7A49E C106S K114R K135Q L142K Y224K E239G His10V250I L253A R306Q R308L E375Y A437G E466K GGGSGGGSGGGSD476H A484E E508R D510R K512Y E515Q K536Q BtagV7 K539Q D570S T571VGGGSGGGSGGGS BtagV7 L44A K135Q K138Q L142K Y224K E239G V250I His10L253A R306Q R308L T368S E375Y A437G E466K GGGSGGGSGGGSD476H A484E E508R D510K K512Y E515Q P558A BtagV7 D570S T571VGGGSGGGSGGGS BtagV7 L142K Y224K E239G V250I L253A R306Q R308L His10E375Y A437G E466K D476H A484E E508R D510K GGGSGGGSGGGSK512Y E515Q P558A D570T T571A BtagV7 GGGSGGGSGGGS BtagV7K138C L142K Y224K E239G V250I L253A R306Q His10R308L E375Y A437G E466K D476H A484E E508R GGGSGGGSGGGSD510K K512Y E515Q D570S T571V BtagV7 GGGSGGGSGGGS BtagV7K138Q L142K Y224K E239G V250I L253A R306Q His10R308L E375Y A437G E466K D476H A484E E508R GGGSGGGSGGGSD510K K512Y E515Q D570S T571V BtagV7 GGGSGGGSGGGS BtagV7K135Q L142K Y224K E239G V250I L253A R306Q His10R308L E375Y A437G E466K D476H A484E E508R GGGSGGGSGGGSD510R K512Y E515Q K539E D570S T571V BtagV7 GGGSGGGSGGGS BtagV7K131E K135Q L142K Y224K E239G V250I L253A His10E375Y A437G D476H A484E E508R D510K K512Y GGGSGGGSGGGSE515Q D523R D570S T571V BtagV7 GGGSGGGSGGGS BtagV7L142K Y224K E239G V250I L253A R306Q R308L His10E375Y A437G E466K D476H A484E E508R D510K GGGSGGGSGGGS K512Y E515Q D570MBtagV7 GGGSGGGSGGGS BtagV7

TABLE 8 Exemplary mutations introduced into an M2Y DNApolymerase. Positions are identified relative to SEQ ID NO: 2.C-terminal region Mutations feature(s) C1035 E236G V247I L250A S253A His10 E372Y A434G A481E V503M E505R GGGSGGGSGGGS D507K K509Y E512QBtagV7

The amino acid sequences of exemplary recombinant Φ29 and M2Ypolymerases harboring the exemplary mutation combinations of Tables 7and 8 are provided in Tables 9 and 10. Table 9 includes the polymeraseportion of the molecule as well as the one or more exogenous features atthe C-terminal region of the polymerase, while Table 10 includes theamino acid sequence of the polymerase portion only.

TABLE 9Amino acid sequences of exemplary recombinant Φ29 and M2Y polymerasesincluding C-terminal exogenous features. Amino acid positions areidentified relative to SEQ ID NO: 1 for recombinant Φ29 polymerases(denoted by ″Phi29″) or relative to SEQ ID NO: 2 for recombinant M2Ypolymerases (denoted by ″M2″). SEQ ID NO Amino Acid Sequence  67MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.Y224K_E239G_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI V2501_L253A_E375Y_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC A437G_A484E_E508R_LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLK D510K_K512Y_E515Q_GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK D570M.G.His10.QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD GGGSGGGSGGGS.BtagV7KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDMTFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE  68MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.A134S_Y224K_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI E239G_V250I_L253A_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC E375Y_A437G_A484E_S487A_LGYKGKRKIHTVIYDSLKKLPFPVKKISKDFKLTVLK E508R_D510K_K512Y_GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK E515Q_I524S_F526L_QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD D570E.G.His10.KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP GGGSGGGSGGGS.BtagV7AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHEATFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDSKLSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE  69MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.L142K_Y224K_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI E239G_V250I_L253A_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC E375Y_A437G_D476H_LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVKK A484E_E508R_D510K_GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK K512Y_L513K_E515Q_QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD D570E.G.His10.KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP GGGSGGGSGGGS.BtagV7.AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF GGGSGGGSGGGS.BtagV7ELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY KVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE  70 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.L142K_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICR306Q_R308L_E375Y_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVKKA437G_D476H_A484E_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKE508R_D510K_K512Y_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDL513K_E515Q_D570E. KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPG.His10.GGGSGGGSGGGS. AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFBtagV7.GGGSGGGSGGGS. ELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSN BtagV7VDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY KVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE  71 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.L142K_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICR306Q_R308L_E375Y_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVKKA437G_E466K_D476H_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKA484E_E508R_D510K_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDK512Y_L513K_E515Q_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP D570E.G.His10.AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF GGGSGGGSGGGS.ELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSN BtagV7.GGGSGGGSGGGS.VDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDK BtagV7WTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY KVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE  72 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.L142K_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICR306Q_R308L_E375Y_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVKKA437G_E466K_D476H_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKA484E_E508R_D510K_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDK512Y_E515Q_D570E. KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPG.His10.GGGSGGGSGGGS. AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFBtagV7.GGGSGGGSGGGS. ELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSN BtagV7VDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE  73 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.L142K_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICE375Y_A437G_E466K_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVKKD476H_A484E_E508R_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKD510K_K512Y_E515Q_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD D570E.G.His10.KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP GGGSGGGSGGGS.AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF BtagV7.GGGSGGGSGGGS.ELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSN BtagV7VDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE  74 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.L142K_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICE375Y_M429A_A437G_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVKKA484E_E508R_D510K_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKK512Y_E515Q_D570E. QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDG.His10.GGGSGGGSGGGS. KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPBtagV7.GGGSGGGSGGGS. AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF BtagV7ELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPAGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE  75 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.A134S_L142K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIY224K_E239G_V250I_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICL253A_E375Y_A437G_ LGYKGKRKIHTVIYDSLKKLPFPVKKISKDFKLTVKKA484E_S487A_E508R_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKD510K_K512Y_E515Q_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDI524S_F526L_D570E.G. KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP His10.AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF GGGSGGGSGGGS.BtagV7ELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHEATFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDSKLSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE  76MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.Y224K_E239G_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI V250I_L253_E375Y_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC A437G_A484E_S487A_LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLK E508R_D510K_K512Y_GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK E515Q_1524S_F526L_QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD D570M.G.His10.KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP GGGSGGGSGGGS.AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF BtagV7ELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHEATFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDSKLSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDMTFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE  77MSRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNY M2.C103S_E236G_V247I_KIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWL L250A_S253A_E372Y_EQHGFKWSNEGLPNTYNTIISKMGQWYMIDISFGYK A434G_A481E_V503MGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDID E505R_D507K_K509YYHTERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLD E512Q.G.His10.RMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIR GGGSGGGSGGGS.BtagV7KAYRGGFTWLNDKYKGKEIGEGMVFDINSAYPAQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGVEPVELYLTNVDLELIQEHYELYNVEYIDGFICFREKTGLFKDFIDKWTYVKTHEYGAKKQLAKLMLNSLYGKFASNPDVTGKVPY LKDDGSLGFRVGDEEYKDPVYTPMGVFITAWGRFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGYLKQCSPDEATTTKFSVKCAGMTDTIKKKVTFDNFAVGFSS MGKPKPVQVNGGVVLVDSVFTIKGHHHHHHHHHHGGGSGGGSGGGSGLNDFFEAQKIEWHE  78 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.L142K_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFID235E_E239G_V250A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICL253H_E375Y_A437G_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVKKA484E_E508R_D510K_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKK512Y_E515Q_D570E. QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDHis10.GGGSGGGSGGGS. KEVRKAYRGGFTWLNERFKGKEIGEGMVFDANSHY BtagV7PAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKG YLVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIKGHHHHHH HHHHGGGSGGGSGGGSGLNDFFEAQKIEWHE  79MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.Y224K_E239G_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI V250I_L253A_E375Y_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC A437G_A484E_E508R_LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLK D510K_K512Y_E515Q.GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK His10.GGGSGGGSGGGS.QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD BtagV7KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE  80MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.V141K_L142K_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI Y224K_E239G_V250I_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC L253A_E375Y_A437G_LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTKKK A484E_E508R_D510K_GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK K512Y_E515Q.His10.QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD GGGSGGGSGGGS.BtagV7KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHE  95MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.K135Q_L142K_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI Y224K_E239G_V250I_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC L253A_R306Q_R308L_LGYKGKRKIHTVIYDSLKKLPFPVKKIAQDFKLTVKK E375Y_A437G_E466K_GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK D476H_A484E_E508R_QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD D510R_K512Y_E515Q_KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP D570S_T571V.G.AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF His10co.GGGSGGGSGGGS.ELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSN BtagV7.VDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDK GGGSGGGSGGGS.BtagV7WTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVRGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDSVFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE  96 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K131E_K135Q_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIL142K_Y224K_E239G_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICV250I_L253A_E375Y_ LGYKGKRKIHTVIYDSLKKLPFPVEKIAQDFKLTVKKA437G_A484E_E508R_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKD510R_K512Y_E515Q_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDD523R_P558A_D570S_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP T571V.G.His10.AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF GGGSGGGSGGGS.ELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSN BtagV7.GGGSGGGSGGGS.VDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDK BtagV7WTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVRGY LVQGSPDDYTRIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKAVQVPGGVVLVDSVFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE  97 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.A49E_C106S_K114R_ HSEYKIGNSLDEFMEWVLKVQADLYFHNLKFDGAFIK131E_K135Q_L142K_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDISY224K_E239G_V250I_ LGYKGKRRIHTVIYDSLKKLPFPVEKIAQDFKLTVKKL253A_Y369E_E375Y_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKA437G_D476H_A484E_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDE508R_D510K_K512Y_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPE515Q_D523R_D570S_ AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF T571V.G.His10.ELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSN GGGSGGGSGGGS.BtagV7.VDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDK GGGSGGGSGGGS.BtagV7WTEIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTRIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDSVFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE  98 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K135Q_K138Q_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIL142K_Y224K_E239G_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICV250I_L253A_R306Q_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAQDFQLTVKKR308L_E375Y_A437G_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKE466K_D476H_A484E_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDE508R_D510K_K512Y_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPE515Q_I524T_P558A_ AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFD570S_T571V.G.His10. ELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSNGGGSGGGSGGGS. VDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDK BtagV7.WTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK GGGSGGGSGGGS.BtagV7VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDTKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKAVQVPGGVVLVDSVFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE  99 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K135Q_K138Q_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIL142K_Y224K_E239G_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICV250I_L253A_R306Q_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAQDFQLTVKKR308L_E375Y_A437G_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKE466K_V475I_D476H_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDA484E_E508R_D510K_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPK512Y_E515Q_I524T_ AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFP558A_D570S_T571V.G. ELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSNHis10.GGGSGGGSGGGS. VDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKBtagV7.GGGSGGGSGGGS. WTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK BtagV7VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIIHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDTKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKAVQVPGGVVLVDSVFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE 100 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K135Q_K138Q_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIL142K_Y224K_E239G_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICV250I_L253A_R306Q_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAQDFQLTVKKR308L_T368S_E375Y_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKA437G_E466K_D476H_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDA484E_E508R_D510K_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPK512Y_E515Q3524T_ AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFP558A_D570S_T571V. ELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSN G.His10.VDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDK GGGSGGGSGGGS.WSYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK BtagV7.GGGSGGGSGGGS.VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGR BtagV7YTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDTKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKAVQVPGGVVLVDSVFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE 101 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K135Q_K138Q_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIL142K_Y224K_E239G_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICV250I_L253A_R306Q_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAQDFQLTVKKR308L_T368S_E375Y_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKA437G_E466K_V475I_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDD476H_A484E_E508R_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPD510K_K512Y_E515Q_ AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFP558A_D570S_T571V. ELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSN G.His10.VDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDK GGGSGGGSGGGS.WSYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK BtagV7.GGGSGGGSGGGS.VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGR BtagV7YTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIIHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKAVQVPGGVVLVDSVFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE 102 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.L44A_K135Q_ HSEYKIGNSADEFMAWVLKVQADLYFHNLKFDGAFIK138Q_L142K_Y224K_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICE239G_V250I_L253A_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAQDFQLTVKKR306Q_R308L_E375Y_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKA437G_E466K_D476H_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDA484E_S487A_E508R_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPD510K_K512Y_E515Q_ AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFP558A_D570S_T571V. ELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSN G.His10.VDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDK GGGSGGGSGGGS.WTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK BtagV7.GGGSGGGSGGGS.VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGR BtagV7YTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHEATFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKAVQVPGGVVLVDSVFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE 103 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K135Q_L142K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIY224K_E239G_V250I_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICL253A_R306Q_R308L_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAQDFKLTVKKT368S_E375Y_A437G_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKE466K_D476H_A484E_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDE508R_D510R_K512Y_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPE515Q_P558A_D570S_ AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF T571V.G.His 10.ELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSN GGGSGGGSGGGS.BtagV7.VDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDK GGGSGGGSGGGS.BtagV7WSYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVRGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKAVQVPGGVVLVDSVFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE 104 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.A49E_C106S_ HSEYKIGNSLDEFMEWVLKVQADLYFHNLKFDGAFIK114R_K135Q_L142K_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDISY224K_E239G_V250I_ LGYKGKRRIHTVIYDSLKKLPFPVKKIAQDFKLTVKKL253A_R306Q_R308L_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKE375Y_A437G_E466K_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDD476H_A484E_E508R_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPD510R_K512Y_E515Q_ AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFK536Q_K539Q_D570S_ ELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSN T571V.G.His10.VDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDK GGGSGGGSGGGS.BtagV7.WTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK GGGSGGGSGGGS.BtagV7VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVRGY LVQGSPDDYTDIKFSVKCAGMTDQIKQEVTFENFKVGFSRKMKPKPVQVPGGVVLVDSVFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE 105 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.L44A_K135Q_ HSEYKIGNSADEFMAWVLKVQADLYFHNLKFDGAFIK138Q_L142K_Y224K_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICE239G_V250I_L253A_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAQDFQLTVKKR306Q_R308L_T368S_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKE375Y_A437G_E466K_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDD476H_A484E_E508R_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPD510K_K512Y_E515Q_ AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFP558A_D570S_T571V. ELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSN G.His10.VDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDK GGGSGGGSGGGS.BtagV7.WSYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK GGGSGGGSGGGS.BtagV7VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKAVQVPGGVVLVDSVFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE 106 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.L142K_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICR306Q_R308L_E375Y_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVKKA437G_E466K_D476H_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKA484E_E508R_D510K_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDK512Y_E515Q_P558A_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP D570T_T571A.G.AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF His10.GGGSGGGSGGGS.ELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSN BtagV7.VDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDK GGGSGGGSGGGS.BtagV7WTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKAVQVPGGVVLVDTAFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE 107 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K138C_L142K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIY224K_E239G_V250I_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICL253A_R306Q_R308L_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFCLTVKKE375Y_A437G_E466K_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKD476H_A484E_E508R_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDD510K_K512Y_E515Q_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPD570S_T571V.G.His10. AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF GGGSGGGSGGGS.ELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSN BtagV7.VDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDK GGGSGGGSGGGS.BtagV7WTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDSVFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE 108 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K138Q_L142K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIY224K_E239G_V250I_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICL253A_R306Q_R308L_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFQLTVKKE375Y_A437G_E466K_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKD476H_A484E_E508R_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDD510K_K512Y_E515Q_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP D570S_T571V.G.AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF His10.GGGSGGGSGGGS.ELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSN BtagV7.VDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDK GGGSGGGSGGGS.BtagV7WTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDSVFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE 109 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K135Q_L142K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIY224K_E239G_V250I_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICL253A_R306Q_R308L_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAQDFKLTVKKE375Y_A437G_E466K_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKD476H_A484E_E508R_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDD510R_K512Y_E515Q_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPK539E_D570S_T571V. AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF G.His10.ELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSN GGGSGGGSGGGS.VDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDK BtagV7.GGGSGGGSGGGS.WTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK BtagV7VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVRGY LVQGSPDDYTDIKFSVKCAGMTDKIKEEVTFENFKVGFSRKMKPKPVQVPGGVVLVDSVFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE 110 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K131E_K135Q_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIL142K_Y224K_E239G_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICV250I_L253A_E375Y_ LGYKGKRKIHTVIYDSLKKLPFPVEKIAQDFKLTVKKA437G_D476H_A484E_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKE508R_D510K_K512Y_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDE515Q_D523R_D570S_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP T571V.G.His10.AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF GGGSGGGSGGGS.ELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSN BtagV7.GGGSGGGSGGGS.VDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDK BtagV7WTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTRIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDSVFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE 111 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.L142K_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICR306Q_R308L_E375Y_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVKKA437G_E466K_D476H_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKA484E_E508R_D510K_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDK512Y_E515Q_D570M. KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP G.His10.AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF GGGSGGGSGGGS.ELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSN BtagV7.GGGSGGGSGGGS.VDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDK BtagV7WTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDMTFTIKGHHHHHHH HHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE

TABLE 10 Amino acid sequences of exemplary recombinant Φ29and M2Y polymerases. Amino acid positions are identifiedrelative to SEQ ID NO: 1 for recombinant Φ29 polymerases(denoted by “Phi29”) or relative to SEQ ID NO: 2 for recombinant M2Y polymerases (denoted by “M2”). SEQ ID NOAmino Acid Sequence 81 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.Y224K_E239G_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIV250I_L253A_E375Y_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICA437G_A484E_E508R_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKD510K_K512Y_E515Q_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK D570MQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDMTFTIK 82 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.A134S_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICE375Y_A437G_A484E_ LGYKGKRKIHTVIYDSLKKLPFPVKKISKDFKLTVLKS487A_E508R_D510K_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKK512Y_E515Q_I524S_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD F526L_D570EKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHEATFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDSKLSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIK 83 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.L142K_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICE375Y_A437G_D476H_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVKKA484E_E508R_D510K_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKK512Y_L513K_E515Q_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD D570EKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY KVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIK 84 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.L142K_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICR306Q_R308L_E375Y_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVKKA437G_D476H_A484E_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKE508R_D510K_K512Y_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDL513K_E515Q_D570E KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY KVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIK 85 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.L142K_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICR306Q_R308L_E375Y_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVKKA437G_E466K_D476H_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKA484E_E508R_D510K_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDK512Y_L513K_E515Q_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP D570EAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY KVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIK 86 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.L142K_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICR306Q_R308L_E375Y_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVKKA437G_E466K_D476H_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKA484E_E508R_D510K_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDK512Y_E515Q_D570E KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIK 87 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.L142K_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICE375Y_A437G_E466K_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVKKD476H_A484E_E508R_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKD510K_K512Y_E515Q_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD D570EKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIK 88 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.L142K_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICE375Y_M429A_A437G_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVKKA484E_E508R_D510K_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKK512Y_E515Q_D570E QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPAGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIK 89 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.A134S_L142K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIY224K_E239G_V250I_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICL253A_E375Y_A437G_ LGYKGKRKIHTVIYDSLKKLPFPVKKISKDFKLTVKKA484E_S487A_E508R_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKD510K_K512Y_E515Q_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDI524S_F526L_D570E KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHEATFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDSKLSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIK 90 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.Y224K_E239G_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIV250I_L253A_E375Y_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICA437G_A484E_S487A_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKE508R_D510K_K512Y_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKE515Q_I524S_F526L_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLD D570MKEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHEATFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDSKLSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDMTFTIK 91 MSRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYM2.C103S_E236G_ KIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWL V247I_L250A_S253A_EQHGFKWSNEGLPNTYNTIISKMGQWYMIDISFGYK E372Y_A434G_A481E_GKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDID V503M_E505R_D507K_YHTERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLD K509Y_E512QRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRKAYRGGFTWLNDKYKGKEIGEGMVFDINSAYPAQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGVEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKDFIDKWTYVKTHEYGAKKQLAKLMLNSLYGKFASNPDVTGKVPY LKDDGSLGFRVGDEEYKDPVYTPMGVFITAWGRFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGYLKQCSPDEATTTKFSVKCAGMTDTIKKKVTFDNFAVGFSS MGKPKPVQVNGGVVLVDSVFTIK 92MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIED Phi29.L142K_Y224K_HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFI D235E_E239G_V250A_INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDIC L253H_E375Y_A437G_LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVKK A484E_E508R_D510K_GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFK K512Y_E515Q_D570EQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNERFKGKEIGEGMVFDANSHYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKG YLVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDETFTIK 93 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.Y224K_E239G_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIV250I_L253A_E375Y_ 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GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKD476H_A484E_E508R_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDD510K_K512Y_E515Q_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP D570S_T571VAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDSVFTIK 125 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K138Q_L142K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIY224K_E239G_V250I_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICL253A_R306Q_R308L_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFQLTVKKE375Y_A437G_E466K_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKD476H_A484E_E508R_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDD510K_K512Y_E515Q_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP D570S_T571VAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDSVFTIK 126 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K135Q_L142K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIY224K_E239G_V250I_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICL253A_R306Q_R308L_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAQDFKLTVKKE375Y_A437G_E466K_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKD476H_A484E_E508R_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDD510R_K512Y_E515Q_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPK539E_D570S_T571V AQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVRGY LVQGSPDDYTDIKFSVKCAGMTDKIKEEVTFENFKVGFSRKMKPKPVQVPGGVVLVDSVFTIK 127 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.K131E_K135Q_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIL142K_Y224K_E239G_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICV250I_L253A_E375Y_ LGYKGKRKIHTVIYDSLKKLPFPVEKIAQDFKLTVKKA437G_D476H_A484E_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKE508R_D510K_K512Y_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDE515Q_D523R_D570S_ KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYP T571VAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTRIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDSVFTIK 128 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDPhi29.L142K_Y224K_ HSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIE239G_V250I_L253A_ INWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICR306Q_R308L_E375Y_ LGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVKKA437G_E466K_D476H_ GDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKA484E_E508R_D510K_ QGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDK512Y_E515Q_D570M KEVRKAYRGGFTWLNDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGY LVQGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDMTFTIK

Additional exemplary polymerase mutations and/or combinations thereofare provided in FIG. 7. Such polymerases can be employed, e.g., innucleic acid amplification, including, e.g., single molecule sequencing.Further exemplary polymerase mutations and/or combinations thereof areprovided in FIG. 8. Such polymerases can be employed, e.g., in nucleicacid amplification, including, e.g., whole genome amplification.Additional exemplary polymerase mutations and/or combinations thereofare provided in FIG. 9. Such polymerases can be employed, e.g., innucleic acid amplification, including, e.g., single molecule sequencingusing large (e.g., protein shield analogs, other shielded analogs, orother analogs with a molecular weight of at least 10,000) analogs. InFIGS. 7-9, positions of the mutations are identified relative to awild-type Φ29 DNA polymerase (SEQ ID NO:1) where the name of thepolymerase includes “Phi29,” and where the name of the polymeraseincludes “M2” positions are identified relative to a wild-type M2Ypolymerase (SEQ ID NO:2). Numbers with a decimal point representinsertions; for example, 96.1 represents an insertion between positions96 and 97. Where the feature “topo V fusion” is listed, it indicatesthat the polymerase includes a fusion as described in de Vega et al.(2010) “Improvement of φ29 DNA polymerase amplification performance byfusion of DNA binding motifs” Proc Natl Acad Sci USA 107:16506-16511.Additional features are listed in Table 11. The notation“pET16.BtagV7co.His10co,” where the tags are listing in the N-terminalposition, indicates that the polymerase includes N-terminal His10 andbiotin tags. The feature “Cterm_His10co” is the same as listing theHis10 in the C terminal position; both terms indicate that thepolymerase includes a C-terminal His10 tag. “pET16” or “pET11” refers toa vector used to produce a recombinant Φ29 polymerase comprising theindicated mutations, and “co” indicates that the polynucleotide sequenceencoding certain features (e.g., a His10 tag or BtagV7) has been codonoptimized; neither notation is relevant to the structure of thepolymerase.

TABLE 11Exemplary exogenous features (e.g., tags, linkers, fusion domains,and the like) that are optionally present in polymerases of theinvention. As noted above, polymerases of the invention can includeany of these features alone or in combination with one or moreadditional features, typically at the N-terminal and/or C-terminalregions of the polymerase. Note that the initial glycine residueshown for the polyhistidine tag is optional. Feature Sequence576GTSGA (linker amino acid GTSGA sequence); SEQ ID NO: 47GGGSGGGSGGGS (linker GGGSGGGSGGGS sequence); SEQ ID NO: 48GGG (linker sequence) GGG GGGS (linker sequence); SEQ ID GGGS NO: 49GGGSGGGS (linker sequence); GGGSGGGS SEQ ID NO: 50GGGSGGGSGGGSGGGS (linker GGGSGGGSGGGSGGGS sequence); SEQ ID NO: 51DYKDDDDK (protease site); SEQ DYKDDDDK ID NO: 52LEVLFQGP (protease site); SEQ ID LEVLFQGP NO: 53ENLYFQG (protease site); SEQ ID ENLYFQG NO: 54 pET16.BtagV7co.His10coMSVDGLNDFFEAQKIEWHEAMGHHHHHH (N-terminal B tag, His tag andHHHHSSGHIEGRH linkers cloned into pET16); SEQ ID  NO: 55G.His10co.GGGSGGGSGGGS.Bta GHHHHHHHHHHGGGSGGGSGGGSGLNDFgV7co or His10co.GGGSGGGSG FEAQKIEWHE GGS.BtagV7co (C-terminal tags andlinkers in polymerases encoded in pET11 vectors); SEQ ID NO: 56G.His10co or His10co (10 histidines GHHHHHHHHHHpreceded by a single amino acid glycine linker; inclusion of theglycine is optional); SEQ ID NO: 57 MeCP2(77-167)co (a portion of(M)ASASPKQRRSIIRDRGPMYDDPTLPEGW MeCpG binding protein 2; the N-TRKLKQRKSGRSAGKYDVYLINPQGKAFR terminal methionine is included inSKVELIMYFEKVGDTSLDPNDFDFTVTGRG cases where this is at the very SPSRbeginning of the polypeptide  chain); SEQ ID NO: 58UL42C-de1ta320.co (HSV UL42 LPRRLHLEPAFLPYSVKAHECCMTDSPGGVconstruct); SEQ ID NO: 59 APASPVEDASDASLGQPEEGAPCQVVLQGAELNGILQAFAPLRTSLLDSLLVMGDRGILIH NTIFGEQVFLPLEHSQFSRYRWRGPTAAFLSLVDQKRSLLSVFRANQYPDLRRVELAITGQ APFRTLVQRIWTTTSDGEAVELASETLMKRELTSFVVLVPQGTPDVQLRLTRPQLTKVLN ATGADSATPTTFELGVNGKFSVFTTSTCVTFAAREEGVSSSTSTQVQILSNALTKAGQAAA NAKTVYGENTHRTFSVVVDDCSMRAVLRRLQVGGGTLKFFLTTPVPSLCVTATGPNAVS AVFLLKPQK SNAPtag (06-alkylguanine DNAMSVDMDKDCEMKRTTLDSPLGKLELSGCE alkyl transferase); SEQ ID NO: 60QGLHEIKLLGKGTSAADAVEVPAPAAVLG GPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISY QQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLAVKEWLLAHEG HRLGKPGLGAMSetAndRingFinger (SRA domain of (M)HMPANHFGPIPGVPVGTMWRFRVQVSEmouse UHRF1; the N-terminal SGVHRPHVAGIHGRSNDGAYSLVLAGGYEmethionine is included in cases DDVDNGNYFTYTGSGGRDLSGNKRTAGQSwhere this is at the very beginning SDQKLTNNNRALALNCHSPINEKGAEAEDof the polypeptide chain); SEQ ID WRQGKPVRVVRNMKGGKHSKYAPAEGN NO: 61RYDGIYKVVKYWPERGKSGFLVWRYLLR RDDTEPEPWTREGKDRTRQLGLTMQYPEGYLEALANKEKSRKR DBJBP1.co (Thymine dioxygenaseTNLMVSTAVEKKKYLDSEFLLHCISAQLLD JBP1 catalytic domain); SEQ IDMWKQARARWLELVGKEWAHMLALNPER NO: 62 KDFLWKNQSEMNSAFFDLCEVGKQVMLGLLGKEVALPKEEQAFWIMYAVHLSAACAE ELHMPEVAMSLRKLNVKLKDFNFGGTRYFKDMPPEEKKRRMERKQRIEEARRHGMP MBP (Maltose binding fusionKIEEGKLVIWINGDKGYNGLAEVGKKFEKD protein); SEQ ID NO: 63TGIKVTVEHPDKLEEKFPQVAATGDGPDIIF WAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKD LLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKD VGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSK VNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPL GAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVD EALKDAQTRITK

The mutations or combinations of mutations shown in FIGS. 7-9 are notlimited to use in a Φ29 or M2Y polymerase. Essentially any of thesemutations, any combination of these mutations, and/or any combination ofthese mutations with the other mutations disclosed or referenced hereincan be introduced into a polymerase (e.g., a Φ29-type polymerase) toproduce a modified recombinant polymerase in accordance with theinvention. Polymerases of the invention including the mutations orcombinations provided in FIGS. 7-9 can include any exogenous orheterologous feature (or combination of such features), e.g., at the N-and/or C-terminal region. Similarly, some or all of the exogenousfeatures listed in FIGS. 7-9 can be omitted, or substituted or combinedwith any of the other exogenous features described herein, and stillresult in a polymerase of the invention. As will be appreciated, thenumbering of amino acid residues is with respect to a particularreference polymerase, such as the wild-type sequence of the Φ29polymerase (SEQ ID NO:1) or M2Y polymerase (SEQ ID NO:2); actualposition of a mutation within a molecule of the invention may vary basedupon the nature of the various modifications that the enzyme includesrelative to the wild type Φ29 enzyme, e.g., deletions and/or additionsto the molecule, either at the termini or within the molecule itself.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. Accordingly, the following examples areoffered to illustrate, but not to limit, the claimed invention.

Example 1: Characterization of Exemplary Recombinant Polymerases inSingle Molecule Sequencing Reactions

Recombinant polymerases based on Φ29 or M2Y polymerase and includingvarious combinations of mutations were expressed and purified asdescribed below. The polymerases were characterized by use in singlemolecule sequencing. Single molecule sequencing data was obtained withrecombinant Φ29 and M2Y polymerases including the mutation combinationslisted in FIG. 7. Exemplary data are presented in Table 12. Data foreach polymerase is presented along with data for a control polymerase,acquired from the same chip for comparison. nReads represents the numberof ZMWs from which single molecule sequencing data was obtained.Accuracy and readlength are determined using data for those readsmeeting selected performance criteria.

TABLE 12Single molecule sequencing with the exemplary recombinant Φ29 and M2Ypolymerases listed in Tables 3-5. Median Median Control Control ReadAccuracy Control Control Median Median % Pol.^(a) nReads length^(b) (%)Pol.^(c) nReads Readlength^(b) Accuracy  7^(e) 82 1371.5 86 C1 199 118985  8^(e) 420 1246.5 85 C2 162 964 86  9^(e) 118 1285.5 86 C3 237 114386 10^(e) 151 1045 85 C3 234 1020 84 11^(e) 150 934.5 88 C4 177 913 8812^(e) 296 505 90 C4 247 944 89 13^(e) 332 871 87 C5 152 805 86 14^(e)244 792 91 C2 260 916.5 91 15^(e) 196 766 90 C2 95 999 90 16^(f) 29731970 89 C6 2654 1835 88 17^(e) 342 1792 81 C3 139 1255 86 18^(d) 2281596 86 N/A N/A N/A N/A 19^(f) 2393 2227 87 C6 2475 2031 87 20^(e) 157994 88 C4 198 938.5 89 21^(f) 2001 3048 86 C7 2284 2561 85 22^(e) 4091011 88 C4 196 966 89 23^(e) 101 247 89 C2 359 1267 90 24^(e) 117 483 87C4 213 967 88 25^(e) 190 405.5 85 C2 139 1204 87 26^(e) 381 744 90 C2129 1278 90 ^(a) SEQ ID NO of exemplary polymerase (see Table 5). ^(b)Median readlength in nucleotides. ^(c) Control polymerases are C1: Φ29K131E L142K Y148I Y224K D235E E239G V250A L253H E375Y A437G A484E E508RD510K K512Y E515Q His10 GGGSGGGSGGGS BtagV7, C2: Φ29 K131E Y224K E239GV250I L253A E375Y A437G A484E D510K K512Y E515Q His10 GGGSGGGSGGGSBtagV7, C3: Φ29 K131E Y148I Y224K D235E E239G L253H E375Y A437G A484ED510K K512Y E515Q His10 GGGSGGGSGGGS BtagV7, C4: Φ29 K131E Y148I Y224KE239G V250I L253A E375Y A437G A484E D510K K512Y E515Q His10 GGGSGGGSGGGSBtagV7, C5: M2Y Y145I E236G V247I L250A S253A E372Y A434G A481E D507KK509Y E512Q His10 GGGSGGGSGGGS BtagV7, C6: Φ29 Q99I K131E A134S Y224KE239G V250I L253A E375Y A437N A484E E508R D510K K512Y E515Q D570E His10GGGSGGGSGGGS BtagV7, and C7: M2Y K128E Y145I E236G V247I L250A S253AE372Y A434G A481E D507K K509Y E512Q His10 GGGSGGGSGGGS BtagV7, wherepositions are identified relative to SEQ ID NO: 1 for C1-C4 and C6 andrelative to SEQ ID NO: 2 for C5 and C7. ^(d) Thepolymerase/primer/template complex was immobilized on the ZMW bottomthrough a biotinylated primer rather than through a biotin tag on thepolymerase as for the other nineteen polymerases in Table 12. No on-chipcontrol polymerase was employed. Data obtained from 20 minute movie.^(e) Data obtained from 7 minute movie. ^(f) Data obtained from 45minute movie.

Materials and Methods

Molecular Cloning

The phi29 and M2Y polymerase genes were cloned into either pET16 orpET11 (Novagen). Primers for specified mutations are designed andintroduced into the gene using the Phusion Hot Start DNA Polymerase Kit(New England Biolabs). A PCR reaction is performed to incorporatemutations and product is purified using ZR-96 DNA Clean andConcentration Kits (Zymo Research). PCR products are digested withNdeI/BamHI and ligated into the vector. Plasmids are transformed intoTOP10 E. coli competent cells, plated on selective media and incubatedat 37° C. overnight. Colonies are selected and plasmid is purified usingQiagen miniprep kits. Plasmids are then sequenced (Sequetech).

Protein Purification

Plasmid containing the recombinant polymerase gene is transformed intoBL21 Star21 CDE3+Biotin Ligase cells (Invitrogen) using heat shock.Transformed cells are grown in selective media overnight at 37° C. 200μL of the overnight culture are diluted into 4 mL of Overnight ExpressInstant TB Medium (EMD Chemicals) supplemented with biotin, glycerol,and antibiotics and grown at 37° C. until controls reach O.D. value of4-6. Cultures are then incubated at 18° C. for 16 hours. Following thisincubation, cells are harvested, resuspended in lysis buffer, and frozenat −80° C. Cells are thawed. The resulting lysate is centrifuged andsupernatant is collected. Polymerase is purified over nickel followed byheparin columns. The resulting proteins are run on gels and quantifiedby SYPRO® staining.

Single Molecule Sequencing

Enzymes are characterized by single molecule sequencing basically asdescribed in Eid et al. (2009) Science 323:133-138 (includingsupplemental information), using reagents similar to those commerciallyavailable in SMRT™ sequencing kits (Pacific Biosciences of California,Inc.). Each enzyme is initially screened with a single 7 minute moviefollowed by secondary screening with 30 minute replicates whereapplicable, or with a single 20 or 45 minute movie. Data presented inTable 12 are from 7, 20, and 45 minute movies as indicated in the table.Enzymes are evaluated, e.g., based on readlength and accuracy comparedto control enzymes.

Example 2: Characterization of Exemplary Recombinant Polymerases inSingle Molecule Sequencing Reactions with Protein Shield Analogs

Recombinant polymerases based on Φ29 or M2Y polymerase and includingvarious combinations of mutations were expressed and purified asdescribed below. The polymerases were characterized by use in singlemolecule sequencing with a set of four protein shield nucleotideanalogs. Single molecule sequencing data was obtained with recombinantΦ29 and M2Y polymerases including the mutation combinations listed inFIG. 9. Exemplary data are presented in Table 13. Data for eachpolymerase is presented along with data for a control polymerase,acquired from the same chip for comparison. nReads represents the numberof ZMWs from which single molecule sequencing data was obtained.Accuracy, readlength, and other characteristics are determined usingdata for those reads meeting selected performance criteria.

TABLE 13 Single molecule sequencing with exemplary recombinant Φ29 andM2Y polymerases listed in Tables 7-9. Control Control Ratio Delta RatioGlobal Ratio Ratio Delta Pol. ^(a) nReads Pol.^(b) nReads RL ^(c) Accur.^(d) Speed ^(e) IPD ^(f) PW ^(g) Pause ^(h) 67 5441 79 4124 1.21 −0.010.99 0.96 0.98 −0.03 68 833 79 4124 0.99 0.01 0.88 1.07 1.14 −0.02 693666 79 2848 1.43 −0.02 1.28 0.63 0.98 −0.03 70 1453 79 1591 1.41 −0.011.39 0.62 0.96 0.01 71 1288 79 1591 1.4 −0.02 1.43 0.59 0.97 0.01 723510 79 1591 1.43 −0.01 1.26 0.74 0.97 0.02 73 3407 79 1591 1.37 −0.011.22 0.72 0.98 −0.01 74 4000 79 1501 1.15 −0.04 0.95 1.05 0.89 −0.03 752371 68 553 1.16 0 1.12 0.94 0.97 0.03 76 645 68 867 1.14 0.01 0.96 0.951.06 −0.03 77 584 79 2113 1.06 −0.01 1.11 0.80 1.08 −0.01 ^(a) SEQ ID NOof exemplary polymerase (see Table 9). ^(b)SEQ ID NO of controlpolymerase (see Table 9). ^(c) Median readlength in nucleotides for thetest polymerase divided by the median readlength for the controlpolymerase ^(d) Median accuracy for the test polymerase minus the medianaccuracy for the control polymerase ^(e) Polymerization rate observedfor the test polymerase divided by the polymerization rate observed forthe control polymerase ^(f) The ratio (test polymerase/controlpolymerase) of the (top 10%) trimmed mean interpulse distance calculatedfor each base and averaged over the four bases ^(g) The ratio (testpolymerase/control polymerase) of the (top 5%) trimmed mean pulse widthcalculated for each base and averaged over the four bases ^(h)[PolRate*(median(PW) + median(IPD))/ln(2)], calculated for the testpolymerase minus that calculated for the control polymerase, wherePolRate is the polymerization rate, PW is pulse width, and IPD isinterpulse distance

Materials and Methods

Molecular Cloning

The phi29 and M2Y polymerase genes were cloned into either pET16 orpET11 (Novagen). Primers for specified mutations are designed andintroduced into the gene using the Phusion Hot Start DNA Polymerase Kit(New England Biolabs). A PCR reaction is performed to incorporatemutations and product is purified using ZR-96 DNA Clean andConcentration Kits (Zymo Research). PCR products are digested withNdeI/BamHI and ligated into the vector. Plasmids are transformed intoTOP10 E. coli competent cells, plated on selective media and incubatedat 37° C. overnight. Colonies are selected and plasmid is purified usingQiagen miniprep kits. Plasmids are then sequenced (Sequetech).

Protein Purification

Plasmid containing the recombinant polymerase gene is transformed intoBL21 Star21 CDE3+Biotin Ligase cells (Invitrogen) using heat shock.Transformed cells are grown in selective media overnight at 37° C. 200μL of the overnight culture are diluted into 4 mL of Overnight ExpressInstant TB Medium (EMD Chemicals) supplemented with biotin, glycerol,and antibiotics and grown at 37° C. until controls reach O.D. value of4-6. Cultures are then incubated at 18° C. for 16 hours. Following thisincubation, cells are harvested, resuspended in lysis buffer, and frozenat −80° C. Cells are thawed. The resulting lysate is centrifuged andsupernatant is collected. Polymerase is purified over nickel followed byheparin columns. The resulting proteins are run on gels and quantifiedby SYPRO® staining.

Single Molecule Sequencing

Enzymes are characterized by single molecule sequencing on a PACBIO™ RSsequencing instrument using standard laser and analysis options,basically as described in Eid et al. (2009) Science 323:133-138(including supplemental information) and Korlach et al. (2010) Methodsin Enzymology 72:431-455, using a set of protein shield analogs with astructure similar to that shown in FIG. 23 of U.S. patent applicationSer. No. 13/767,619 corresponding to bases A, G, C, and T. Each of theprotein shield nucleotide analogs has 6 phospholinked nucleotidemoieties linked to a streptavidin through a bis-biotin linker. Each ofthe nucleotide analogs has a different dye component. The T analog has adouble dye moiety with an emission maximum of about 558 nm, the G analoghas a FRET dye pair with an emission maximum of about 598 nm, the Aanalog has a single dye with an emission maximum of about 659 nm, andthe C analog has a FRET dye pair with an emission maximum of about 697nm. Other reagents are similar to those commercially available in SMRT™C2 sequencing kits (Pacific Biosciences of California, Inc.) with theaddition of 650 nM free streptavidin in the chip wash buffer. Eachenzyme is screened with a single 90 minute movie. Enzymes are evaluated,e.g., based on readlength and accuracy compared to control enzymes.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

What is claimed is:
 1. A composition comprising: a recombinant DNApolymerase, which recombinant polymerase comprises an amino acidsequence that is at least 80% identical to SEQ ID NO:1 or at least 80%identical to SEQ ID NO:2, and which recombinant polymerase comprises oneor more mutations selected from the group consisting of an amino acidsubstitution at position A435, an amino acid substitution at position1452, an A83E substitution, an A83R substitution, and a K539Esubstitution, wherein identification of positions is relative to SEQ IDNO:1.
 2. The composition of claim 1, wherein the recombinant polymerasecomprises one or more mutations selected from the group consisting of anA435S substitution and an I452F substitution, wherein identification ofpositions is relative to SEQ ID NO:1.
 3. The composition of claim 1,wherein the recombinant polymerase further comprises one or moremutations selected from the group consisting of an amino acidsubstitution at position 224, an amino acid substitution at position239, an amino acid substitution at position 253, an amino acidsubstitution at position 375, an amino acid substitution at position437, an amino acid substitution at position 484, an amino acidsubstitution at position 510, an amino acid substitution at position512, and an amino acid substitution at position 515, whereinidentification of positions is relative to SEQ ID NO:1.
 4. Thecomposition of claim 3, wherein the recombinant polymerase comprises oneor more mutations selected from the group consisting of a Y224Ksubstitution, an E239G substitution, an L253A substitution, an E375Ysubstitution, an A437G substitution, an A437N substitution, an A484Esubstitution, a D510K substitution, a K512Y substitution, and an E515Qsubstitution, wherein identification of positions is relative to SEQ IDNO:1.
 5. The composition of claim 3, wherein the recombinant polymerasecomprises E375Y, A484E, and K512Y substitutions, wherein identificationof positions is relative to SEQ ID NO:1.
 6. The composition of claim 1,wherein the recombinant polymerase comprises an amino acid sequence thatis at least 90% identical to SEQ ID NO:1.
 7. The composition of claim 1,wherein the recombinant polymerase comprises an amino acid sequence thatis at least 90% identical to SEQ ID NO:2.
 8. The composition of claim 1,wherein the recombinant polymerase comprises one or more exogenousfeatures at the C-terminal and/or N-terminal region of the polymerase.9. The composition of claim 8, wherein the recombinant polymerasecomprises a biotin ligase recognition sequence and a polyhistidine tag.10. The composition of claim 1, wherein the recombinant polymerasecomprises two tandem biotin ligase recognition sequences at theC-terminal region of the polymerase.
 11. The composition of claim 1,comprising a phosphate-labeled nucleotide analog.
 12. The composition ofclaim 11, wherein the nucleotide analog comprises a fluorophore.
 13. Thecomposition of claim 1, comprising a phosphate-labeled nucleotide analogand a DNA template, wherein the recombinant polymerase incorporates thenucleotide analog into a copy nucleic acid in response to the DNAtemplate.
 14. The composition of claim 1, wherein the composition ispresent in a DNA sequencing system.
 15. The composition of claim 14,wherein the sequencing system comprises a zero-mode waveguide.
 16. Thecomposition of claim 15, wherein the recombinant polymerase isimmobilized on a surface of the zero-mode waveguide in an active form.17. A method of sequencing a DNA template, the method comprising: a)providing a reaction mixture comprising: the DNA template, a replicationinitiating moiety that complexes with or is integral to the template,the recombinant DNA polymerase of claim 1, wherein the polymerase iscapable of replicating at least a portion of the template using themoiety in a template-dependent polymerization reaction, and one or morenucleotides and/or nucleotide analogs; b) subjecting the reactionmixture to a polymerization reaction in which the recombinant polymerasereplicates at least a portion of the template in a template-dependentmanner, whereby the one or more nucleotides and/or nucleotide analogsare incorporated into the resulting DNA; and c) identifying a timesequence of incorporation of the one or more nucleotides and/ornucleotide analogs into the resulting DNA.
 18. The method of claim 17,wherein the subjecting and identifying steps are performed in a zeromode waveguide.
 19. A method of making a DNA, the method comprising: (a)providing a reaction mixture comprising: a template, a replicationinitiating moiety that complexes with or is integral to the template,the recombinant DNA polymerase of claim 1, which polymerase is capableof replicating at least a portion of the template using the moiety in atemplate-dependent polymerase reaction, and one or more nucleotidesand/or nucleotide analogs; and (b) reacting the mixture such that thepolymerase replicates at least a portion of the template in atemplate-dependent manner, whereby the one or more nucleotides and/ornucleotide analogs are incorporated into the resulting DNA.
 20. Themethod of claim 19, wherein the mixture is reacted in a zero modewaveguide.
 21. The method of claim 19, the method comprising detectingincorporation of the one or more nucleotides and/or nucleotide analogs.